Polypeptide with reduced allergenicity

ABSTRACT

The invention relates to modified polypeptides with reduced allergenicity comprising a parent polypeptide with a molecular weight from between 10 kDa and 100 kDa conjugated to a polymer with a molecular weight (M r ) in the range of 1 kDa and 60 kDa. The modified polypeptide are produced using a process including the step of conjugating from 1 to 30 polymer molecules with the parent polypeptide. Further the invention relates to compositions comprising said polypeptides and further ingredients normally used in e.g. detergents, including dishwashing detergents and soap bars, household article, agrochemicals, personal care products, cosmetics, toiletries, oral and dermal pharmaceuticals, composition for treating textiles, and compositions used for manufacturing food and feed. Finally the invention is directed to uses of polypeptides with reduced allergenicity or compositions thereof for reducing the allergenicity of products for a vast number of industrial applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/150,891,filed Sep. 10, 1998, now U.S. Pat. No. 5,981,718; which is acontinuation of application Ser. No. 08/836,293 filed May 12, 1997 nowU.S. Pat. No. 5,856,451, which is a continuation of PCT/DK95/00497,filed Dec. 7, 1995; and claims priority under 35 U.S.C. 119 of Danishapplications having Ser. Nos. 1395/94, 1396/94, 1397/94, 1398/94,1399/94, 1400/94, and 1401/94, all of which were filed on Dec. 7, 1994;the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to modified polypeptides, such as proteins orenzymes, with reduced allergenicity. Further the invention is directedtowards a process for producing said modified polypeptides with reducedallergenicity, and also compositions thereof. Finally the inventionrelates to uses of said modified polypeptides with reduced allergenicityor compositions thereof.

BACKGROUND OF THE INVENTION

An increasing number of polypeptides, including proteins and enzymes,are being produced industrially by microorganisms for use in industry,household, food/feed, cosmetics or medicine etc. Said polypeptides mayunder certain circumstances inflict a potential risk to especiallyemployees handling the manufacturing of products containingpolypeptides, and also to some extent to users of these products, suchas hairdressers, and end-users of cosmetic and toiletry products etc.

This potential risk need to be controlled and/or limited.

Allergenicity of Polypeptides

In general polypeptides are potential antigens toward which the humanimmune system can produce specific antibodies upon exposure. Thisprocess is known as "immunization" when a clinical beneficial responseis obtained whereas the term "sensitization" is applied when theresponse leads to hypersensitivity. During the primary exposure clonalselection and expansion of the specific B-cell clones are initiated,meaning that a protective or allergic response will only be a clinicallymanifest upon following exposures. The allergic reaction can be definedas an pathological immune response elicited by otherwise unharmfulagents in low concentrations.

The process of sensitisation leading to type IV hypersensitivity arecharacterized by the formation of specific IgE antibodies. At present,the mechanism controlling the subclass shifting are not fullyunderstood.

IgE secreted from activated B-cells can attach to Fcε receptors locatedon the surface of mast cells and basophil granulocytes, which containnumerous cytoplasmic granules packed with chemical mediators e.g.histamine (J. Klein, "Immunology", Blackwell Sci. Pub., London, 1990; E.Benjamini & S. Leskowitz, "Immunology", Wiley-Liss, N.Y. 1991).

In atopic individuals each of these cells can have a high number of IgEmolecules bound to its surface, where they can remain available tointeract with allergens for weeks. Upon contact with an allergen thesurface bound IgE crossbinds the allergen, leading to the release ofcytoplasmic granules into the proximity of the cell, thereby causing theinflammatoric allergic reaction.

The role of IgE has been shown to relate to natural immunologic defencesystems towards parasitic worms infections and the development ofallergies has been suggested to be an unfortunate by-product of thisdefence system.

The natural allergens causing IgE mediated hypersensitivity can beclassified according to their way of exposure: Inhalant allergens(pollens, dust mites etc.), Ingested allergens (milk, eggs etc.);Contact allergens (e.g. from latex) and allergens from stinging insects(e.g. bees, fire ants etc.). The aero-allergens represents clinically byfar the largest group, stressing an area of high potential risk for theindustrial polypeptides.

Testing for allergy can either be performed as in vivo provocation, mostcommonly skin prick testing of by a number of in vitro assays, primarilybased on IgE levels in pheriperal blood. In spite of great efforts inthe latter area the most reliable way to diagnose allergy is still thein vivo challenging, which again has different levels of sensitivitydepending on the selected target organ.

For instance, intranasal challenge with allergenic proteins can provokean allergic response even though skin tests and radioallergosorbent test(RAST) for specific serum IgE are negative (Ivan Roitt, "EssentialImmunology", fifth edition, p. 152 and p. 240, 1984).

Reduction of Allergenicity of Polypeptides

Presently, the generation of allergic responses to industrialpolypeptides are avoided by immobilizing, granulating, coating ordissolving the products, especially to avoid the formation of airbornematerial. Anyhow, these methods still represent a risk of dust oraerosol formation during handling and processing, with the subsequentrisk of allergic sensitisation.

There will anyhow still be a risk of having polypeptide dust ordissolved polypeptide in aerosol form. Therefore some release of enzymescan occur leading to a possible sensitisation and subsequent allergicresponse.

Another way of diminishing the problem has been to select polypeptidesof human origin for production, e.g. in bacteria, fungi, yeast, ormammalian cell cultures. This may alleviate some problems for humans,but not for animals. Furthermore, it will in many cases not be possibleto find polypeptides of human origin with the desired properties,wherefore other origin has to be considered. This can be either humanpolypeptides that are altered in one or more positions in the molecule,giving the performance that is desired. It might also be molecules fromother species, including bacteria, mold etc. All the latter groups ofproducts will have potency for immune stimulation in mammalians.

A further proposition for decreasing allergenicity has been to reducethe size of the protein molecules (see e.g. JP Patent Publication No.4,112,753, or Research Disclosure No. 335,102). This is, however, asolution that is only available when the activity of the protein iswithout importance, or in such rare cases, where the activity of theprotein is retained in spite of a breakdown of the protein.

The application of protein engineering has been suggested to reduce theallergenicity of proteins through epitope mapping and subsequent changeof the allergenic epitopes (see WO 92/10755 (Novo Nordisk A/S). Thisprocedure usually requires a large investment in work and development.

In the medicinal field suggestions have been made of diminishing theimmunogenicity of polypeptides through the attachment of polymermolecules to the polypeptide. This usually has the effect of interferingwith the interactions of the polypeptide with other macromolecularstructures. Such a conjugate may also exhibit novel properties: e.g. EP38 154 (Beecham Group Ltd.) discloses conjugates of allergens withpolysarcosine which have immunosuppressive properties.

U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenicpolypeptides, such as enzymes and peptide hormones coupled topolyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100moles of polymer are used per mole polypeptide and at least 15% of thephysiological activity is maintained. In addition the clearance time incirculation is prolonged, due to the increased size of the PEG-conjugateof the polypeptides in question. The protected polypeptide is injectedin an aqueous solution into the mammalian circulatory system orintramuscular. The immunogenicity is assessed from intradermal injectiontests.

U.S. Pat. No. 4,179,337 concerns therapeutic applications and theretaining of the corresponding physiological activity. In the context oftherapeutic applications it is important to limit the risk of inflictingimmunological responses caused by exposure of the allergensintradermally, intravenously or subcutaneously. However controllingrespiratory allergens are of no importance. Furthermore the relativeamount of polymer necessary to conjugate the polypeptides makes themethod expensive.

WO 93/15189 (Veronese et al.) concerns a method to maintain the activityin polyethylene glycol-modified proteolytic enzymes by linking theproteolytic enzyme to a macromolecularized inhibitor. The conjugates areintended for medical applications

It has been found that the attachment of polypeptides to polymers ingeneral has the effect of reducing the activity of the polypeptide orinterfering with the interaction between the polypeptide and itssubstrate. EP 183 503 (Beecham Group PLC) discloses a development of theabove concept by providing conjugates comprising pharmaceutically usefulproteins linked to at least one water-soluble polymer by means of areversible linking group.

GB patent No. 1,183,257 (Crook et al.) describes chemistry forconjugation of enzymes to polysaccharides via a triazine ring.

EP 471 125 (Kanebo, LTD.) describes a modified protease linked to apolysaccharide via a triazine ring leading to a suppressing effect onantigenicity and dermal hypersensitivity. The employed polysaccharidehas an average molecular weight not less than 10 kDa. The modificationrate for surface amino acid groups in the modified protease is not lessthat 30%.

In general it is believed that allergens, entering the respiratorytract, must have a molecular weight below about 100 kDa in order topenetrate the plasma membrane and cause allergic reactions.

Folkeson et al., Acta Physiol. Scand, 139, p. 437-354, 1990, showed thatthere is an inverse relationship between the molecular weight of aninstilled protein marker and the transferred amount (bioavailability)via the respiratory tract to the blood stream.

WO 94/10191 (Novo Nordisk A/S) discloses a process for production of lowallergenic protein, wherein the monomeric parent protein molecules arelinked together to form an oligomer. This is done e.g. by using a linkeror spacer molecule or by linking the monomeric molecules together bypeptide bonds between the C-terminal of the first monomer and theN-terminal of the second monomer.

EP 215 662 (Masda, Hiroshi) concerns a modified or unmodified proteasederived from microorganisms for use in medicaments such as anti-tumouragent. It is suggested that the modification of the protease may becarried out by e.g. coupling with a saccharide, introduction of ahydrophobic polymeric group, conjugation with a low molecular weightanti-tumour agent of a molecular weight less than 2 kDa.

Activation of Polymers

Methods and chemistry for activation of polymers as well as forconjugation of proteins are intensively described in the literature.Commonly used methods for activation of insoluble polymers includeactivation of functional groups with cyanogen bromide, periodate,glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone,carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F.Taylor, (1991), "Protein immobilisation. Fundamental and applications",Marcel Dekker, N.Y.; S. S. Wong, (1992), "Chemistry of Proteinconjugation and Crosslinking", CRC Press, Boca Raton; G. T. Hermanson etal., (1993), "Immobilized Affinity Ligand Techniques", Academic Press,N.Y.). Some of the methods concern activation of insoluble polymers butare also applicable to activation of soluble polymers e.g. periodate,trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc.The functional groups being amino, hydroxyl, thiol, carboxyl, aldehydeor sulfydryl on the polymer and the chosen attachment group on theprotein must be considered in choosing the activation and conjugationchemistry which normally consist of i) activation of polymer, ii)conjugation, and iii) blocking of residual active groups.

Several reviews and monographs (Harris, (1985), JMS-REV. Macronol. Chem.Phys. C25, 325-373; Scouten, (1987), Methods in Enzymology vol. 135,Mosbach, K., Ed., Academic Press: Orlando, 30-65; Wong et al., (1992),Enzyme Microb. Technol., 14, 866-874; Delgado et al., (1992), CriticalReviews in Therapeutic Drug Carrier Systems, 9, 249-304; Zalipsky,(1995), Bioconjugate Chem., 6, 150-165) have been made concerning thesynthesis of activated polyethylene glycols (PEGs).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540 (Enzon), and U.S. Pat.No. 5,281,698 (Cetus), and more WO 93/15189 (Veronese) and forconjugation between activated polymers and enzymes e.g. CoagulationFactor VIII (WO 94/15625), haemoglobin (WO 94/09027), oxygen carryingmolecule (U.S. Pat. No. 4,412,989), ribonuclease and superoxidedismutase (Veronese at al., App. Biochem. Biotech., 11, p. 141-45,1985).

THE OBJECT OF PRIOR ART

The relevant prior art concern reducing the immunological response orhypersensitivity (allergy) of polypeptides, proteins and/or enzymes inapplications for therapeutic purposes, which is relevant when presentingthe allergens intradermally, intravenously or subcutaneously. Howeverprior art do not concern presentation of allergens in industrialapplications, which potentially may inflict allergy when inhaled, or inapplications, where the end-user may be exposed to polypeptides, forinstance, in the use of detergents, cleaning preparations, toiletriesand other Personal Care products.

It would therefore be desirable to be able to provide polypeptides,proteins and/or enzymes for non-therapeutic industrial applications witha reduced allergic response when inhaled, and which polypeptidessubstantially maintain their catalytic activity.

SUMMARY OF THE INVENTION

The present inventors have now surprisingly provided modifiedpolypeptides with reduced allergenicity substantially maintaining thecatalytic activity.

First of all the invention relates to modified polypeptide with reducedallergenicity Comprising a parent polypeptide with a molecular weight ofbetween 10 kDa and 100 kDa conjugated to a polymer with a molecularweight in the range of 1 kDa and 60 kDa.

In a preferred embodiment of the invention-the polypeptide is a proteinor an enzyme.

The invention is also directed towards a process for producing saidppolypeptide with reduced allergenicity comprising the step ofconjugating from 1 to 30 polymer molecules to a parent polypeptide.

Further the invention provides compositions comprising said polypeptideand/or other enzymes/polypeptides and/or ingredients normally used ine.g. detergents, including dishwashing detergents and soap bars,household articles, agrochemicals, personal care products, includingcleaning preparations e.g. for contact lenses, cosmetics, toiletries,oral and dermal pharmaceuticals, composition for treating textiles, andcompositions used for manufacturing food, e.g. for baking, and feed.

Finally the invention relates to uses of polypeptides, proteins orenzymes with reduced allergenicity or compositions thereof for a vastnumber of industrial applications.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 shows a sectional view of a rat to be intratracaeally exposed toenzymes.

FIG. 2 shows the specific IgE response in Brown Norway Rats sera tomodified Subtilisin Novo and parent Subtilisin Novo.

FIG. 3 shows the specific IgE response in Brown Norway Rats sera tomodified Lipolase® and parent Lipolase®.

FIG. 4 shows the;specific IgE response in Brown Norway Rats sera tomodified Laccase and parent Laccase.

FIG. 5 shows the specific IgE response in Brown Norway Rats sera tomodified Carezyme® and parent Carezyme®.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have now surprisingly succeeded in providingmodified polypeptides with reduced allergenicity, wherein the catalyticactivity is at least substantially maintained.

These modified polypeptides with reduced allergenicity according to theinvention solve some of the previously mentioned problems that allergensmay inflict when inhaled, within a number of non-therapeuticapplications.

Regarding non-therapeutic applications it must be emphasized that it ismainly inhalation of the allergens that may inflict the risk of allergicresponses.

Therefore, it is to be understood that one of the crucial advantages ofthe invention is that the inventors have solved one of the majorproblems of polypeptides within a vast number of industrialapplications, as inhalation, including intratracheal and intranasalpresentation of allergens is the main problem in the context ofallergenicity. In contrast hereto prior art solutions mainly concerntherapeutic applications where intradermally, intravenously orsubcutaneously presentation of the allergens are the main problem.Further, inhalation of allergens is a much more sensitive question.

Normally when discussing production for therapeutic purposes it concernsproduction of enzymes in kilogram scale, while production for industrialpurposes concerns production of many 1000 kilograms. Techniques used fortherapeutic purposes can not always advantageously be adapted forindustrial purposes.

The term "reduced allergenicity" indicates that the amount of producedIgE (in humans, and molecules with comparable effects in specificanimals), which can lead to an allergic state, is decreased wheninhaling a modified polypeptides of the invention in comparison to thecorresponding parent polypeptides.

The terms "immunogen", "antigen" and "allergen" are defined below. Theterm "immunogen" is the wider term and includes "antigen" and"allergen".

An "immunogen" may be defined as a substance which when introduced intoanimals, including humans, is capable of stimulating an immunologicresponse.

The term "antigen" refers to substances which by themselves are capableof generating antibodies when recognized as a non-self molecule.

Further, an "allergen" may be defined as an antigen which may give riseto allergic sensitization or an allergic response by IgE antibodies (inhumans, and molecules with comparable effects in animals).

As mentioned above it is, in the context of enzymes includingpolypeptides for industrial applications, important to distinguishbetween allergens mediating allergic responses e.g. intradermally, andrespiratory allergens causing allergic responses by contact withcell-bound IgE in the respiratory tract, due to the fact thatintradermal tests may be negative even though inhalation tests provokean allergic response.

Therefore, assessment of allergenicity may be made by inhalation tests,comparing the effect of intratracheally (into the trachea) administratedparent polypeptides with the corresponding modified polypeptide withreduced allergenicity according to the invention.

A number of in vitro animal models exist for assessment of theallegenicity of polypeptides. Some of these models give a suitable basisfor hazard assessment in man. Suitable models include a guniea pig modeland a rat model. These models seek to identify respiratory allergens asa function of elicitation reactions induced in previously sensitisedanimals. According to these models the alleged allergens are introducedintratracheally into the animals.

A suitable strain of guinea pigs, the Dunkin Hartley strain, do not ashumans, produce IgE antibodies in connection with the allergic response.However, they produce another type of anti-body the IgG1A and IgG1B (seee.g. PrentΦ, ATLA, 19, p. 8-14, 1991), which are responsible for theirallergenic response to inhaled polypeptides including enzymes. Thereforewhen using the Dunkin Hartley animal model, the relative amount of IgG1Aand IgG1B is a measure of the allergenicity level.

A rat strain suitable for intratracheal exposure to polypeptides andenzymes is the Brown Norway strain. The Brown Norway strain produces IgEas the allergic response.

In Example 22 the surprising discoveries of the present invention isdisclosed showing that the allergenicity of polypeptides, in thespecific cases enzymes, can be reduced by increasing the weight of theenzyme by e.g. coupling a number of polymers to the polypeptidemolecule.

Other animals such as rabbits etc. may also be used for comparablestudies.

In the first aspect the invention is directed towards modifiedpolypeptides with reduced allergenicity comprising a parent polypeptideswith a molecular weight of between 10 kDa and 100 kDa conjugated to apolymer with a molecular weight in the range of 1 kDa and 60 kDa.

The Parent Polypeptide

According to the invention the parent polypeptide may be any polypeptidefor industrial applications. This include proteins, enzymes,anti-microbial polypeptides, ligands, inhibitors, enhancers andco-factors.

In a preferred embodiment of the invention parent polypeptide is anenzyme and may be selected from the group of enzymes mentioned in thefollowing.

Parent Proteases

Parent proteases (i.e. enzymes classified under the EnzymeClassification number E.C. 3.4 in accordance with the Recommendations(1992) of the International Union of Biochemistry and Molecular Biology(IUBMB)) include proteases within this group.

Examples include proteases selected from those classified under theEnzyme Classification (E.C.) numbers: 3.4.11 (i.e. so-calledaminopeptidases) including 3.4.11.5 (Prolyl aminopeptidase), 3.4.11.9(X-pro aminopeptidase), 3.4.11.10 (Bacterial leucyl aminopeptidase),3.4.11.12 (Thermophilic aminopeptidase), 3.4.11.15 (Lysylaminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18(Methionyl aminopeptidase).

3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1(Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.25

(Cucumisin), 3.4.21.32 (Brachyurin), 3.4.21.43 (Cerevisin) and 3.4.21.62(Subtilisin);

3.4.22 (i.e. so-called cysteine endopeptidases), including 3.4.22.2(Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7(Asclepain), 3.4.22.14 (Actinidain), 3.4.22.30 (Caricain) and 3.4.22.31(Ananain);

3.4.23 (i.e. so-called aspartic endopeptidases), including 3.4.23.1(Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin)and 3.4.23.25 (Saccharopepsin); and

3.4.24 (i.e. so-called metalloendopeptidases), including 3.4.24.28(Bacillolysin).

Examples of relevant subtilisins comprise subtilisin BPN', subtilisinamylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase,subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147,thermitase, aqualysin, Bacillus PB92 protease, proteinase K, ProteaseTW7, and Protease TW3.

Specific examples of such readily available commercial proteases includeEsperase®, Alcalase®, Neutrase®, Dyrazym®, Savinase®, Pyrase®,Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro, Clear-Lens Pro (allenzymes available from Novo Nordisk A/S).

Examples of other commercial proteases include Maxtase®, Maxacal®,Maxapem® marketed by Gist-Brocades N.V., Opticlean® marketed by Solvayet Cie. and Purafect® marketed by Genencor International.

It is to be understood that also protease variants are contemplates asthe parent protease. Examples of such protease variants are disclosed inEP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomaset al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol.Biol.,

193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Nova Nordisk A/S),WO 91/00345 (Nova Nordisk A/S), EP 525 610 (Solvay) and WO 94/02618(Gist-Brocades N.V.).

The activity of proteases can be determined as described in "Methods ofEnzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol.5.

Parent Lipases

Parent lipases (i.e. enzymes classified under the Enzyme Classificationnumber E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)) include lipases within this group.

Examples include lipases selected from those classified under the EnzymeClassification (E.C.) numbers:

3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (3.1.1.3)Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase A₂.

Examples of lipases include lipases derived from the followingmicroorganisms. The indicated patent publications are incorporatedherein by reference:

Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var. thermoideaand H. insolens (U.S. Pat. No. 4,810,414)

Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps.fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat.No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps.pseudoalcaligenes (EP 218 272) or Ps. mendocina (WO 88/09367; U.S. Pat.No. 5,389,536).

Fusarium, e.g. F. oxysporum (EP 130,064) or F. solani pisi (WO90/09446).

Mucor (also called Rhizomucor), e.g. M. miehei (EP 238 023).

Chromobacterium (especially C. viscosum) Aspergillus (especially A.niger).

Candida, e.g. C. cylindracea (also called C. rugosa) or C. antarctica(WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO89/02916).

Geotricum, e.g. G. candidum (Schimada et al., (1929), J. Biochem., 106,383-388)

Penicillium, e.g. P. camembertii (Yamaguchi et al., (1991), Gene 103,61-67).

Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R.niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) orR. oryzae.

Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica etBiophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992)or B. pumilus (WO 91/16422).

Specific examples of readily available commercial lipases includeLipolase®, Lipolase™ Ultra, Lipozyme®, Palatase®, Novozym® 435,Lecitase® (all available from Novo Nordisk A/S).

Examples of other lipases are Lumafast™, Ps. mendocian lipase fromGenencor Int. Inc.; Lipomax™, Ps. pseudoalcaligenes lipase from GistBrocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) fromUnilever; Bacillus sp. lipase from Solvay enzymes. Other lipases areavailable from other companies.

It is to be understood that also lipase variants are contemplated as theparent enzyme. Examples of such are described in e.g. WO 93/01285 and WO95/22615.

The activity of the lipase can be determined as described in "Methods ofEnzymatic Analysis", Third Edition, 1984, Verlag Chemie, Weinhein, vol.4, or as described in AF 95/5 GB (available on request from Novo NordiskA/S).

Parent Oxidoreductases

Parent oxidoreductases (i.e. enzymes classified under the EnzymeClassification number E.C. 1 (Oxidoreductases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)) include oxidoreductases within this group.

Examples include oxidoreductases selected from those classified underthe Enzyme Classification (E.C.) numbers: Glycerol-3-phosphatedehydrogenase [NAD+] (1.1.1.8), Glycerol-3-phosphate dehydrogenase[NAD(P)⁺ ] (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase [NADP](1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5),Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alaninedehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamatedehydrogenase [NAD(P)⁺ ] (1.4.1.3), Glutamate dehydrogenase [NADP⁺ ](1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase(1.4.1.7), Valine dehydrogenase [NADP⁺ ] (1.4.1.8), Leucinedehydrogenase (1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acidoxidase (1.4.3.2.), 3-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase(1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase(1.4.3.14), L-Aspartate oxidase (1.4.3.16), D-amino-acid dehydrogenase(1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase(1.6.4.5), Protein disulfide reductase (glutathione) (1.8.4.2), Laccase(1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase(1.13.11.12), Superoxide dismutase (1.15.1.1):

Said Glucose oxidases may be derived from Aspergillus niger.

Said Laccases may be derived from Polyporus pinsitus, Myceliophtorathermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctoniapraticola, Scytalidium thermophilum and Rhus vernicifera.

Bilirubin oxidases may be derived from Myrothechecium verrucaria.

The Peroxidase may be derived from e.g. Soy bean, Horseradish orCoprinus cinereus.

The Protein Disulfide reductase may be any mentioned in any of the DKpatent applications no. 768/93, 265/94 and 264/94 (Novo Nordisk A/S),which are herby incorporated as reference, including Protein Disukfidereductases of bovine origin, Protein Disulfide reductases derived fromAspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived fromEscherichia coli.

Specific examples of readily available commercial oxidoreductasesinclude Gluzyme™ (enzyme available from Novo Nordisk A/S). However,other oxidoreductases are available from others.

It is to be understood that also variants of oxidoreductases arecontemplated as the parent enzyme.

The activity of oxidoreductases can be determined as described in"Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie,Weinheim, vol. 3.

Parent Carbohydrases

Parent carboydrases may be defined as all enzymes capable of breakingdown carbohydrate chains (e.g. starches) of especially five and sixmember ring structures (i.e. enzymes classified under the EnzymeClassification number E.C. 3.2 (glycosidases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)). Also included in the group of carbohydrasesaccording to the invention are enzymes capable of isomerizingcarbohydrates e.g. six member ring structures, such as D-glucose to e.g.five member ring structures like D-fructose.

Examples include carbohydrases selected from those classified under theEnzyme Classification (E.C.) numbers:

α-amylase (3.2.1.1) β-amylase (3.2.1.2), glucan 1,4-α-glucosidase(3.2.1.3), cellulase (3.2.1.4), endo-1,3(4)-β-glucanase (3.2.1.6),endo-1,4-β-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase(3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17),β-glucosidase (3.2.1.21), α-galactosidase (3.2.1.22), β-galactosidase(3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-β-xylosidase(3.2.1.37), glucan endo-1,3-β-D-glucosidase (3.2.1.39), α-dextrinendo-1,6-glucosidase (3.2.1.41), sucrose α-glucosidase (3.2.1.48),glucan endo-1,3-α-glucosidase (3.2.1.59), glucan 1,4-β-glucosidase(3.2.1.74), glucan endo-1,6-β-glucosidase (3.2.1.75), arabinanendo-1,5-α-arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase(3.2.1.132) and xylose isomerase (5.3.1.5).

Examples of relevant carbohydrases include α-1,3-glucanases derived fromTrichoderma harzianum; α-1,6-glucanases derived from a strain ofPaecilomyces; β-glucanases derived from Bacillus subtilis; β-glucanasesderived from Humicola insolens; β-glucanases derived from Aspergillusniger; β-glucanases derived from a strain of Trichoderma; β-glucanasesderived from a strain of Oerskovia xanthineolytica;exo-1,4-α-D-glucosidases (glucoamylases) derived from Aspergillus niger;α-amylases derived from Bacillus subtilis; α-amylases derived fromBacillus amyloliquefaciens; α-amylases derived from Bacillusstearothermophilus; α-amylases derived from Aspergillus oryzae;α-amylases derived from non-pathogenic microorganisms; α-galactosidasesderived from Aspergillus niger; Pentosanases, xylanases, cellobiases,cellulases, hemi-cellulases deriver from Humicola insolens; cellulasesderived from Trichoderma reesei; cellulases derived from non-pathogenicmold; pectinases, cellulases, arabinases, hemi-celluloses derived fromAspergillus niger; dextranases derived from Penicillium lilacinum;endo-glucanase derived from non-pathogenic mold; pullulanases derivedfrom Bacillus acidopullyticus; β-galactosidases derived fromKluyveromyces fragilis; xylanases derived from Trichoderma reesei;

Specific examples of readily available commercial carbohydrases includeAlpha-Gal™, Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™Plus, Novozyme® 188, Carezyme®, Celluclast®, Cellusoft®, Ceremyl®,Citrozym™, Denimax™, Dezyme™, Dextrozyme™, Finizym®, Fungamyl™,Gamanase™, Glucanex®, Lactazym®, Maltogenase™, Pentopan™, Pectinex™,Promozyme®, pulpzyme™, Novamyl™, Termamyl®, AMG (Amyloglucosidase Novo),maltogenase®, Sweetzyme®, Aquazym® (all enzymes available from NovaNordisk A/S). Other carbohydrases are available from other companies.

It is to be understood that also carbohydrase variants are contemplatedas the parent enzyme.

The activity of carbohydrases can be determined as described in "Methodsof Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim,Vol. 4.

Parent Transferases

Parent transferases (i.e. enzymes classified under the EnzymeClassification number E.C. 2 in accordance with the Recommendations(1992) of the International Union of Biochemistry and Molecular Biology(IUBMB)) include transferases within this group.

The parent transferases may be any transferase in the subgroups oftransferases: transferases transferring one-carbon groups (E.C. 2.1);transferases transferring aldehyde or residues (E.C 2.2);acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4);transferases transferring alkyl or aryl groups, other that methyl groups(E.C. 2.5); transferases transferring nitrogeneous groups (2.6).

In a preferred embodiment the parent transferease is a transglutaminaseE.C 2.3.2.13 (Protein-glutamine μ-glutamyltransferase).

Transglutaminases are enzymes capable of catalyzing an acyl transferreaction in which a gamma-carboxyamide group of a peptide-boundglutamine residue is the acyl donor. Primary amino groups in a varietyof compounds may function as acyl acceptors with the subsequentformation of monosubstituted gamma-amides of peptide-bound glutamicacid. When the epsilon-amino group of a lysine residue in apeptide-chain serves as the acyl acceptor, the transferases formintramolecular or intermolecular gamma-glutamyl-epsilon-lysylcrosslinks.

Examples of transglutaminases are described in the pending DK patentapplication no. 990/94 (Novo Nordisk A/S).

The parent transglutaminase may the of human, aminal (e.g. bovine) ormicrobially origin.

Examples of such parent transglutaminases are animal derivedTransglutaminase, FXIIIa; microbial transglutaminases derived fromPhysarum polycephalum (Klein et al., Journal of Bacteriology, Vol. 174,p. 2599-2605); transglutaminases derived from Streptomyces sp.,including Streptomyces lavendulae, Streptomyces lydicus (formerStreptomyces libani) and Streptoverticillium sp., includingStreptoverticillium mobaraense, Streptoverticillium cinnamoneum, andStreptoverticillium griseocarneum (Motoki et al., U.S. Pat. No.5,156,956; Andou et al., U.S. Pat. No. 5,252,469; Kaempfer et al.,Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al.,International Journal of Sytematic Bacteriology, Vol. 44, p. 285-292;Andou et al., U.S. Pat. No. 5,252,469; Williams et al., Journal ofGeneral Microbiology, Vol. 129, p. 1743-1813).

It is to be understood that also transferase variants are contemplatedas the parent enzyme.

The activity of transglutaminases can be determined as described in"Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie,Weinheim, vol. 1-10.

Parent Phytases

Parent phytases are included in the group of enzymes classified underthe Enzyme Classification number E.C. 3.1.3 (Phosphoric MonoesterHydrolases) in accordance with the Recommendations (1992) of theInternational Union of Biochemistry and Molecular Biology (IUBMB)).

Phytases are enzymes produced by microorganisms which catalyse theconversion of phytate to inositol and inorganic phosphorus

Phytase producing microorganisms comprise bacteria such as Bacillussubtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomycescerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum,Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus orAspergillus nidulans, and various other Aspergillus species).

Examples of parent; phytases include phytases selected from thoseclassified under the Enzyme Classification (E.C.) numbers: 3-phytase(3.1.3.8) and 6-phytase (3.1.2.26). The activity of phytases can bedetermined as described in "Methods of Enzymatic Analysis", thirdedition, 1984, Verlag Chemie, Weinheim, vol. 1-10, or may be measuredaccording to the method described in EP-A1-0 420 358, Example 2 A.

Parent Antimicrobial Polypeptides

Parent anti-microbial polypeptides may be any polypeptides exhibitinganti-microbial activities, such as anti-fungal, anti-bacterial, and/oranti-insecticidal activity.

Said polypeptides may also exhibit other activities such as enzymaticactivity.

Examples of parent anti-microbial polypeptides according to theinvention include: fungicidally active polypeptides derived from themold genus Curvularia described in WO 94/01459 (Novo Nordisk A/S);anti-bacterial polypeptides described in EP 403.458 (Kabigen AB);anti-microbial proteins isolated from the Mirabilis seed, descriped inWO 92/15691 (Imperial Chem Ind. PLC); anti-bacterial polypeptidesisolated from an extract of pig small intestine, described in WO92/22578 (Boman et al.); polypeptide with yeast lethal actionaccumulated by yeast of Hansenula spp. as descriped in JP-60130599;Phytolacca insularis antiviral protein, which can be used as ananti-microbial described in U.S. Pat. No. 5,348,865 (Jin Ro LTD.);bacteriolytic enzymes preparations derived from Nocardiopsisdassonvillei described in U.S. Pat. No. 5,354,681 (Novo Industri A/S).

Examples of other anti-microbial polypeptides are maganinin, protegrin,defensin, pseudomycin, mutanolysin and N-acetylmuramidase.

Relevant parent polypeptides, proteins or enzymes according to theinvention are polypepeptides, proteins or enzymes that may causeallergic reactions. These polypeptides, proteins or enzymes are believedto have a molecular weight between 10 kDa and 100 kDa, preferablybetween 15 kDa and 80 kDa, or between 20 kDa and 70 kDa, or between 25kDa and 60 kDa, or between 28 kDa and 55 kDa, or between 30 kDa and 50kDa.

It is within the scope of the present invention to use variants havingadditional attachment groups, such as amino-groups, in comparison to theparent enzyme. It is advantageous to use such variants as such variantsmore effectively shield the enzyme towards recognition by the immunesystem.

The Polymer

Examples of suitable polymers include polymers selected from the groupcomprising polyalkylene oxides (PAO), such as polyalkylene glycols(PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols(mPEG) and polypropylen glycols, PEG-glycidyl ethers (Epox-PEG),PEG-oxycarbonylimidazole (CDI-PEG), Star-PEGs, Branced PEGs, polyvinylalcohol (PVA), poly-carboxylates, poly-(vinylpyrolidone), poly-D,L-aminoacids, dextrans including carboxymethyldextrans, celluloses, includingmethylcellulose, carboxymethylcellulose, ethylcellulose,hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose,hydrolysates of chitosan, starches such as hydroxyethyl-straches andhydroxypropyl-starches, glycogen, agaroses and derivates thereof, guargum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acidhydrolysates and bio-polymers.

Star PEGs are multi-armed PEG molecules made by polymerization of ethyloxide molecules from a crosslinked divinyl benzene core (Gnanou et al.,(1988), Makromol. Chem 198, 2885; Rein wt al., (1993), Acta Polymer, 44,225). Star PEGS and Branced PEGs are available from Shearwater Inc.,USA).

Epox-PEGs (or PEG-glycidyl ether) are PEGs with an epoxide as theactivated coupling group ar the end. They can undergoreactions/attachment with amino-, hydroxyl- and thiol-groups ofproteins/Elling and Kula, (1991), Biotech. Appl. Biochem, 13, 354).Epox-PEGs are available from Shearwater Inc., USA, as e.g.methoxy-PEG-epoxides and PEG-(epoxides)₂.

CDI-PEGs (or PEG oxycarbonylimidazole) are PEGs with a carbonylimodazoleas the reactive end-group. Said reactive/activated attachment groupconjugates with the protein via a urethane linkage (Beauchamp et al.,1993), Anal. Biochem., 131, 125). CDI-PEGs are available from ShearwaterInc., USA, as e.g. methoxy-PEG-CDI and PEG-(CDI)₂.

Examples of such suitable readily available polymer products (of whichsome are activated polymers) include polyethylene glycols (e.g. fromMerck) having an average molecular weight of between about 1 kDa and 35kDa, methoxypolyethylene glycols (e.g. from Sigma) having an averagemolecular weight of about 5 kDa and dextrans (e.g. from Fluka) having anaverage molecular weight of between about 1 kDa and 60 kDa and evenhigher.

Even though all of the above mentioned polymers can be used according tothe invention the methoxypolyethylene glycols may advantageously beused. This arise from the fact that methoxyethylene glycols only haveone reactive end capable of conjugating with the polypeptide.Consequently, the risk of cross linking is less pronounced. Further, itmakes the product more homogeneous and the reaction of the polymer withthe polypeptide easier to control.

Polymers having a molecular weight (M_(r)) between 1 and 60 kDa may beused according to the invention. Preferred are polymers having amolecular weight (M_(r)) of between 2 kDa and 35 kDa, especially between2 kDa and 25 kDa, such as about 5 kDa or about 15 kDa.

Note that all polymer molecular weights mentioned in this applicationare average molecular weights.

In a preferred embodiment of the invention the polymer is a polyethyleneglycol (PEG), such as a methoxypolyethylene glycol (mPEG).

Activation of Polymers

If the polymer to be used for conjugating the polypeptide is not activeit must be activated by a suitable method. The methods referred in the"Background of the Invention" section are examples of methods which maybe used according to the present invention. However, the most suitablemethod may differ for molecule to molecule dependent on e.g. availableattachment groups on the polypeptide chain.

In the following further methods of suitable polymer activation methodswill be described shortly. However, it is to be understood that alsoother methods can be used.

Coupling polymers to the free acid groups of enzymes can be performedwith the aid of diimide and for example amino-PEG or hydrazino-PEG(Pollak et al., (1976), J. Amr. Chem. Soc., 98, 289-291) ordiazoacetate/amide (Wong et al., (1992), supra).

Coupling polymers to hydroxy groups are generally very difficult as itmust be performed in water. Usually hydrolysis predominates overreaction with hydroxyl groups.

Coupling polymers to free sulfhydryl groups can be reached with specialgroups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone(U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference forsulfhydryl groups but is not as selective as the other mentioned.

Accessible arginine residues in the polypeptide chain may be targeted bygroups comprising two vicinal carbonyl groups.

Techniques involving coupling electrophilically activated PEGs to theamino groups of lysins can also be useful. Many of the usual leavinggroups for alcohols give rise to an amine linkage. For instance, alkylsulfonates, such as tresylates (Nilsson et al., (1984), Methods inEnzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p.56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach,K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987),Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press:Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc.1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris etal., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), arylsulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively convertshydroxy groups in a number of polymers, e.g. PEG, into good leavinggroups (sulfonates) that, when reacted with nucleophiles like aminogroups in polypeptides allow stable linkages to be formed betweenpolymer and polypeptide. In addition to high conjugation yields, thereaction conditions are in general mild (neutral or slightly alkalinepH, to avoid denaturation and little or no disruption of activity), andsatisfy the non-destructive requirements to the polypeptide.

Tosylate is more reactive than the mesylate but also more unstabledecomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995),supra). Epoxides may also been used for creating amine bonds but aremuch less reactive than the above mentioned groups.

Converting PEG into a chloroformate with phosgene gives rise tocarbamate linkages to lysins. This theme can be played in many variantssubstituting the chlorine with N-hydroxy succinimide (U.S. Pat. No.5,122,614, (1992), Zalipsky; Zalipsky et al., (1992), Biotechnol. Appl.Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem.,6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.)etc. The derivatives are usually made by reacting the chloroformate withthe desired leaving group. All these groups give rise to carbamatelinkages to the peptide.

Furthermore, isocyanates and isothiocyanates may be employed yieldingureas and thioureas, respectively.

Amides may be obtained from PEG acids using the same leaving groups asmentioned above and cyclic imid thrones (U.S. Pat. No. 5,349,001,(1994), Greenwald et al.). The reactivity of these compounds are veryhigh but may make the hydrolysis to fast.

PEG succinate made from reaction with succinic anhydride can also beused. The hereby comprised ester group make the conjugate much moresusceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky).This group may be activated with N-hydroxy succinimide.

Furthermore, a special linker can be introduced. The oldest beingcyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252,3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al.,(1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

Coupling of PEG to an aromatic amine followed by diazotation yields avery reactive diazonium salt which in situ can be reacted with apeptide. An amide linkage may also be obtained by reacting an azlactonederivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.)thus introducing an additional amide linkage.

As some peptides do not comprise many lysins it may be advantageous toattach more than one PEG to the same lysine. This can be done e.g. bythe use of 1,3-diamino-2-propanol.

PEGs may also be attached to the amino-groups of the polypeptide withcarbamate linkages (WO 95/11924 Greenwald et al.). Lysine residues mayalso be used as the backbone.

The Conjugate

According to the invention conjugates of modified polypeptide have atotal molecular weight in the range from 50 kDa to 500 kDa, preferably50 kDa to 400, more preferred 50 kDa to 250 kDa, especially 100 kDa to250 kDa, such as 80 kDa to 200 kDa.

A modified polypeptide according to the invention may demonstrate a highdegree of stability.

For most applications, including Personal Care applications, themodified enzymes may advantageously be irreversible conjugated to thepolymer, which entails that the product has only negligible tendency todisintegrate, which would lead to the return of conditions that maycause an allergenic state.

However, in certain other cases it is advantageous that the enzymes stayconjugated to the polymer in the production and/or bulk handling phase,but disintegrates later on, when the enzyme do not inflict a risk ofexposure to humans or animals.

The disintegration of the conjugated modified polypeptide of theinvention may be activated e.g. by physical conditions, such as pH,ionic strength, temperature, reduction or oxidation potential etc. Anexample of this is disintegration upon dissolving a detergentformulation.

Further, the presence of specific compounds may result indisintegration, e.g. into molecules being less conjugated and/ormolecules in the parent form.

Especially in the case where the activity of the polypeptide, protein orenzyme is reduced in the conjugated form, disintegration may beadvantageous.

The invention also relates to a process for producing polypeptides withreduced allergenicity comprising the step of conjugating the parentpolypeptide with from 1 to 30 polymer molecules, preferably 1 to 25,such as 1 to 10 polymer molecules.

Examples of said polymers which may be used according to the inventionare listed above.

Preferably between 1 and 25 polymer molecules are conjugated to eachpolypeptide molecule. This is less than corresponding prior arttechniques. Consequently the expense to polymer is reduced. To someextent it entails that the activity of the polypeptide, protein orenzyme is substantially maintained, as it is to be anticipated that theactivity vary inversely with the number and the size of polymerconjugated to the polypeptide chain.

According to the invention more than 5%, in most cases about 20% to 50%,better 50% to 70%, even better between 70% and 80%, up to between 80%and 90% and even up to 100%, of the activity of the polypeptide ismaintained.

Composition

The invention also relates to a composition comprising at least onepolypeptide, protein or enzyme of the invention.

The composition may further comprise other polypeptides, proteins orenzymes and/or ingredients normally used in e.g. detergents, includingsoap bars, household articles, agrochemicals, personal care products,such as cleaning preparations e.g. for contact lenses, cosmetics,toiletries, oral and dermal pharmaceuticals, composition use fortreating textiles, compositions used for manufacturing food, e.g.baking, and feed etc.

Examples of said polypeptides/proteins/enzymes include enzymesexhibiting protease, lipase, oxidoreductase, carbohydrase, transferase,such as transglutaminase, phytase and/or anti-microbial polypeptideactivity. These enzymes may be present as conjugates with reducesactivity.

It is also contemplated according to the invention to combine the use ofconjugated enzymes with the same activity having different specificity.

Detergent Compositions

If the polypeptide of the invention is an enzyme it may typically beused in detergent composition. It may be included in the detergentcomposition in the form of a non-dusting granulate, a stabilized liquid,or a protected enzyme. Non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to NovoIndustri A/S) and may optionally be coated by methods known in the art.Examples of waxy coating materials are poly(ethylene oxide) products(polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in patent GB 1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Other enzyme stabilizersare well known in the art. Protected enzymes may be prepared accordingto the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g. as powder,granules, paste or liquid. A liquid detergent may be aqueous, typicallycontaining up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as e.g. proteases, amylases, lipases, cutinases,cellulases, peroxidases, oxidases, and further anti-microbialpolypeptides.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂ O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

The detergent composition of the invention comprising the polypeptide ofthe invention may be stabilized using conventional stabilizing agents,e.g. a polyol such as propylene glycol or glycerol, a sugar or sugaralcohol, lactic acid, boric acid, or a boric acid derivative such as,e.g., an aromatic borate ester, and the composition may be formulated asdescribed in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

Particular forms of detergent compositions within the scope of theinvention include:

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                           7-12%                                                  (calculated as acid)                                                          alcohol ethoxysulfate  1-4%                                                   (e.g. C.sub.12-18  alcohol, 1-2 EO) or                                        alkyl sulfate (e.g. C.sub.16-18)                                              alcohol ethoxylate  5-9%                                                      (e.g. C.sub.14-15  alcohol, 7 EO)                                             sodium carbonate (as Na.sub.2 CO.sub.3) 14-20%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  2-6%                            zeolite (as NaAlSiO.sub.4) 15-22%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  0-6%                                   sodium citrate/citric acid  0-15%                                             (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                sodium perborate (as NaBO.sub.3.H.sub.2 O) 11-18%                             TAED  2-6%                                                                    carboxymethylcellulose  0-2%                                                  polymers (e.g. maleic/acrylic acid  0-3%                                      copolymer, PVP, PEG)                                                          enzymes  0-5%                                                                 minor ingredients (e.g. suds  0-5%                                            suppressors, perfume, optical                                                 brightener, photobleach)                                                    ______________________________________                                    

2) A detergent composition formulated as a granulate having a 40 bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                             6-11%                                                (calculated as acid)                                                          alcohol ethoxysulfate  1-3%                                                   (e.g. C.sub.12-18  alcohol, 1-2 EO)                                           or alkyl sulfate (e.g. C.sub.16-18)                                           alcohol ethoxylate  5-9%                                                      (e.g. C.sub.12-18  alcohol, 7 EO)                                             sodium carbonate (as Na.sub.2 CO.sub.3) 15-21%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  1-4%                            zeolite (as NaAlSiO.sub.4) 24-34%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  4-10%                                  sodium citrate/citric acid  0-15%                                             (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                carboxymethylcellulose  0-2%                                                  polymers (e.g. maleic/acrylic acid copolymer,  1-6%                           PVP, PEG)                                                                     enzymes  0-5%                                                                 minor ingredients  0-5%                                                       (e.g. suds suppressors, perfume)                                            ______________________________________                                    

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesultonate                                                                             5-9%                                                 (calculated as acid)                                                          alcohol ethoxylate  7-14%                                                     (e.g. C.sub.12-15  alcohol, 7 EO)                                             soap as fatty acid  1-3%                                                      (e.g. C.sub.16-22  fatty acid)                                                sodium carbonate (as Na.sub.2 CO.sub.3) 10-17%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  3-9%                            zeolite (as NaAlSiO.sub.4) 23-33%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  0-4%                                   sodium perborate (as NaBO.sub.3.H.sub.2 O)  8-16%                             TAED  2-8%                                                                    phosphonate (e.g. EDTMPA)  0-1%                                               carboxymethylcellulose  0-2%                                                  polymers (e.g. maleic/acrylic acid copolymer,  0-3%                           PVP, PEG)                                                                     enzymes  0-5%                                                                 minor ingredients (e.g. suds suppressors,  0-5%                               perfume, optical brightener)                                                ______________________________________                                    

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        linear alkylbenzenesultonate                                                                             8-12%                                                (calculated as acid)                                                          alcohol ethoxylate 10-25%                                                     (e.g. C.sub.12-15  alcohol, 7 EO)                                             sodium carbonate (as Na.sub.2 CO.sub.3) 14-22%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  1-5%                            zeolite (as NaAlSiO.sub.4) 25-35%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  0-10%                                  carboxymethylcellulose  0-2%                                                  polymers (e.g. maleic/acrylic acid copolymer,  1-3%                           PVP, PEG)                                                                     enzymes  0-5%                                                                 minor ingredients (e.g. suds suppressors,  0-5%                               perfume, optical brightener)                                                ______________________________________                                    

5) An aqueous liquid detergent composition comprising

    ______________________________________                                        alcohol ethoxylate      12-18%                                                  (e.g. C.sub.12-15  alcohol, 7 EO)                                             C.sub.12-15  alcohol, 5 EO)                                                   soap as fatty acid (e.g. oleic acid)  3-13%                                   alkenylsuccinic acid (C.sub.12-14)  0-13%                                     aminoethanol  8-18%                                                           citric acid  2-8%                                                             phosphonate  0-3%                                                             polymers (e.g. PVP, PEG)  0-3%                                                borate (as B.sub.4 O.sub.7)  0-2%                                             ethanol  0-3%                                                                 propylene glycol  8-14%                                                       enzymes  0-5%                                                                 minor ingredients  0-5%                                                       (e.g. dispersants, suds suppressors,                                          perfume, optical brightener)                                                ______________________________________                                    

6) An aqueous structured liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                            15-21%                                                (calculated as acid)                                                          alcohol ethoxysulfate  3-9%                                                   (e.g. C.sub.12-18  alcohol, 7 EO),                                            or C.sub.12-15  alcohol, 5 EO)                                                soap as fatty acid (e.g. oleic acid)  3-10%                                   zeolite (as NaAlSiO.sub.4) 14-22%                                             potassium citrate  9-18%                                                      borate (as B.sub.4 O.sub.7)  0-2%                                             carboxymethylcellulose  0-2%                                                  polymers (e.g. PEG, PVP)  0-3%                                                anchoring polymers such as,  0-3%                                             e.g., lauryl methacrylate/acrylic acid copolymer;                             molar ratio 25:1; MW 3800                                                     glycerol  0-5%                                                                enzymes  0-5%                                                                 minor ingredients  0-5%                                                       (e.g. dispersants, suds suppressors,                                          perfume, optical brighteners)                                               ______________________________________                                    

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        fatty alcohol sulfate      5-10%                                                ethoxylated fatty acid monoethanolamide  3-9%                                 soap as fatty acid  0-3%                                                      sodium carbonate (as Na.sub.2 CO.sub.3)  5-10%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  1-4%                            zeolite (as NaAlSiO.sub.4) 20-40%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  2-8%                                   sodium perborate (as NaBO.sub.3.H.sub.2 O) 12-18%                             TAED  2-7%                                                                    polymers (e.g. maleic/acrylic acid copolymer,  1-5%                           PEG)                                                                          enzymes  0-5%                                                                 minor ingredients (e.g. optical brightener,  0-5%                             suds suppressors, perfume)                                                  ______________________________________                                    

8) A detergent composition formulated as a granulate comprising

    ______________________________________                                        ethoxylated fatty acid monoethanolamide                                                                 5-11%                                                 soap as fatty acid  0-3%                                                      sodium carbonate (as Na.sub.2 CO.sub.3)  4-10%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  1-4%                            zeolite (as NaAlSiO.sub.4) 30-50%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  3-11%                                  sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)  5-12%                   polymers (e.g. PVP,  1-5%                                                     maleic/acrylic acid copolymer, PEG)                                           enzymes  0-5%                                                                 minor ingredients (e.g. suds suppressors,  0-5%                               perfume)                                                                    ______________________________________                                    

9) A detergent composition formulated as a granulate comprising

    ______________________________________                                        nonionic surfactant,      1-4%                                                  soap as fatty acid  2-6%                                                      sodium carbonate (as Na.sub.2 CO.sub.3) 14-22%                                zeolite (as NaAlSiO.sub.4) 18-32%                                             sodium sulfate (as Na.sub.2 SO.sub.4)  5-20%                                  sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)  3-8%                    sodium perborate (as NaBO.sub.3.H.sub.2 O)  4-9%                              bleach activator (e.g. NOBS or TAED)  1-5%                                    carboxymethylcellulose  0-2%                                                  polymers (e.g. polycarboxylate or PEG)  1-5%                                  enzymes  0-5%                                                                 minor ingredients  0-5%                                                       (e.g. optical brightener, perfume)                                          ______________________________________                                    

10) An aqueous liquid detergent composition comprising

    ______________________________________                                        alcohol ethoxysulfate     8-15%                                                 (e.g. C.sub.12-15  alcohol, 2-3 EO)                                           alcohol ethoxylate  3-9%                                                      (e.g. C.sub.12-15  alcohol, 7 EO)                                             or C.sub.12-15  alcohol, 5 EO)                                                soap as fatty acid (e.g. lauric acid)  0-3%                                   aminoethanol  1-5%                                                            sodium citrate  5-10%                                                         hydrotrope: (e.g. sodium toluenesulfonate)  2-6%                              borate (as B.sub.4 O.sub.7)  0-2%                                             carboxymethylcellulose  0-1%                                                  ethanol  1-3%                                                                 propylene glycol  2-5%                                                        enzymes  0-5%                                                                 minor ingredients (e.g. polymers,  0-5%                                       dispersants, perfume, optical brighteners)                                  ______________________________________                                    

11) An aqueous liquid detergent composition comprising

    ______________________________________                                        linear alkylbenzenesulfonate                                                                            20-32%                                                (calculated as acid)                                                          alcohol ethoxylate  6-12%                                                     (e.g. C.sub.12-15  alcohol, 7 EO,                                             C.sub.12-15  alcohol, 5 EO,                                                   aminoethanol  2-6%                                                            citric acid  8-14%                                                            borate (as B.sub.4 O.sub.7)  1-3%                                             polymer (e.g. maleic/acrylic acid copolymer,  0-3%                            anchoring plymer such as, e.g.,                                               lauryl methacrylate/acrylic acid                                              copolymer)                                                                    glycerol  3-8%                                                                enzymes  0-5%                                                                 minor ingredients (e.g. hydrotropes,  0-5%                                    dispersants, perfume, optical brighteners)                                  ______________________________________                                    

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        anionic surfactant (linear                                                                              25-40%                                                alkylbenzenesulfonate, alkyl sulfate, alpha-                                  olefinsulfonate, alpha-sulfo fatty acid                                       methyl esters, alkanesulfonates, soap)                                        nonionic surfactant  1-10%                                                    (e.g. alcohol ethoxylate)                                                     sodium carbonate (as Na.sub.2 CO.sub.3)  8-25%                                soluble silicates (as Na.sub.2 O, 2SiO.sub.2)  5-15%                          sodium sulfate (as Na.sub.2 SO.sub.4)  0-5%                                   zeolite (as NaAlSiO.sub.4) 15-28%                                             sodium perborate (as NaBO.sub.3.4H.sub.2 O)  0-20%                            bleach activator (TAED or NOBS)  0-5%                                         enzymes  0-5%                                                                 minor ingredients  0-3%                                                       (e.g. perfume, optical brighteners)                                         ______________________________________                                    

13) Detergent formulations as described in 1)-12) wherein all or part ofthe linear alkylbenzenesulfonate is replaced by (C₁₂ -C₁₈) alkylsulfate.

14) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        (C.sub.12 -C.sub.18) alkyl sulfate                                                                      9-15%                                                 alcohol ethoxylate  3-6%                                                      polyhydroxy alkyl fatty acid amide  1-5%                                      zeolite (as NaAlSiO.sub.4) 10-20%                                             layered disilicate (e.g. 10-20%                                               SK56 from Hoechst)                                                            sodium carbonate (as Na.sub.2 CO.sub.3)  3-12%                                soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  0-6%                            sodium citrate  4-8%                                                          sodium percarbonate 13-22%                                                    TAED  3-8%                                                                    polymers (e.g. polycarboxylates and PVP)  0-5%                                enzymes  0-5%                                                                 minor ingredients (e.g. optical brightener,  0-5%                             photo bleach, perfume, suds suppressors)                                    ______________________________________                                    

15) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        (C.sub.12 -C.sub.18) alkyl sulfate                                                                      4-8%                                                  alcohol ethoxylate 11-15%                                                     soap  1-14%                                                                   zeolite MAP or zeolite A 35-45%                                               sodium carbonate (as Na.sub.2 CO.sub.3)  2-8%                                 soluble silicate (as Na.sub.2 O, 2SiO.sub.2)  0-4%                            sodium percarbonate 13-22%                                                    TAED  1-8%                                                                    carboxymethyl cellulose  0-3%                                                 polymers (e.g. polycarboxylates and PVP)  0-3%                                enzymes  0-5%                                                                 minor ingredients (e.g. optical  0-3%                                         brightener, phosphonate, perfume)                                           ______________________________________                                    

16) Detergent formulations as described in 1)-15) which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

17) Detergent compositions as described in 1), 3), 7), 9) and 12)wherein perborate is replaced by percarbonate.

18) Detergent compositions as described in 1), 3), 7), 9), 12) 14) and15) which additionally contain a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds bleaching", Nature, 369, p.637-639, 1994.

19) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g. phosphate), enzymeand alkali. The detergent may also comprise anionic surfactant and/or ableach system.

The enzyme of the invention may be incorporated in concentrationsconventionally employed in detergents. It is at present contemplatedthat, in the detergent composition of the invention, the enzyme inquestion with reduced allergenicity may be added in an amountcorresponding to 0.001-100 mg of enzyme per liter of wash liquor.

Dishwashing Composition

Further, a modified enzyme according to the invention may also be usedin dishwashing detergents.

Dishwashing detergent compositions comprise a surfactant which may beanionic, non-ionic, cationic, amphoteric or a mixture of these types.The detergent will contain 0-90% of non-ionic surfactant such as low- tonon-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts ofinorganic and/or organic types. The detergent builders may be subdividedinto phosphorus-containing and non-phosphorus-containing types. Thedetergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders,when present, include the water-soluble salts especially alkali metalpyrophosphates, orthophosphates, and polyphosphates. An example ofphosphorus-containing organic alkaline detergent builder, when present,includes the water-soluble salts of phosphonates. Examples ofnon-phosphorus-containing inorganic builders, when present, includewater-soluble alkali metal carbonates, borates and silicates as well asthe various types of water-insoluble crystalline or amorphous aluminosilicates of which zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammoniumand substituted ammonium, citrates, succinates, malonates, fatty acidsulphonates, carboxymetoxy succinates, ammonium polyacetates,carboxylates, polycarboxylates, amino-polycarboxylates, polyacetylcarboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weightpolymers and co-polymers known to have builder properties, for exampleappropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acidcopolymers and their salts.

The dishwashing detergent composition may contain bleaching agents ofthe chlorine/bromine-type or the oxygen-type. Examples of inorganicchlorine/bromine-type bleaches are lithium, sodium or calciumhypochlorite and hypobromite as well as chlorinated trisodium phosphate.Examples of organic chlorine/bromine-type bleaches are heterocyclicN-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water-solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable.

The oxygen bleaches are preferred, for example in the form of aninorganic persalt, preferably with a bleach precursor or as a peroxyacid compound. Typical examples of suitable peroxy bleach compounds arealkali metal perborates, both tetrahydrates and monohydrates, alkalimetal percarbonates, persilicates and perphosphates. Preferred activatormaterials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilizedusing conventional stabilizing agents for the enzyme(s), e.g. a polyolsuch as e.g. propylene glycol, a sugar or a sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition of the invention may also containother conventional detergent ingredients, e.g. deflocculant material,filler material, foam depressors, anti-corrosion agents, soil-suspendingagents, sequestering agents, anti-soil redeposition agents, dehydratingagents, dyes, bactericides, fluorescers, thickeners and perfumes.

Finally, the enzyme of the invention may be used in conventionaldishwashing detergents, e.g. in any of the detergents described in anyof the following patent publications: EP 518719, EP 518720, EP 518721,EP 516553, EP 516554, EP 516555, GB 2200132, DE 3741617, DE 3727911, DE4212166, DE 4137470, DE 3833047, WO 93/17089, DE 4205071, WO 52/09680,WO 93/18129, WO 93/04153, WO 92/06157, WO 92/08777, EP 429124, WO93/21299, U.S. Pat. No. 5141664, EP 561452, EP 561446, GB 2234980, WO93/03129, EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, U.S.Pat. No. 5112518, EP 318204, EP 318279, EP 271155, EP 271156, EP 346136,GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197, U.S. Pat. No.5240632.

Particular forms of dishwashing detergent compositions within the scopeof the invention include:

    ______________________________________                                        1) POWDER AUTOMATIC DISHWASHING COMPOSITION                                   ______________________________________                                        Nonionic surfactant     0.4-2.5%                                                Sodium metasilicate 0-20%                                                     Sodium disilicate 3-20%                                                       Sodium triphosphate 20-40%                                                    Sodium carbonate 0-20%                                                        Sodium perborate 2-9%                                                         Tetraacetylethylenediamine (TAED) 1-4%                                        Sodium sulphate 5-33%                                                         Enzymes including modified enzymes 0.0001-0.5%                              ______________________________________                                    

    ______________________________________                                        2) POWDER AUTOMATIC DISHWASHING COMPOSITION                                   ______________________________________                                        Nonionic surfactant     1-2%                                                    (e.g. alcohol ethoxylate)                                                     Sodium disilicate 2-30%                                                       Sodium carbonate 10-50%                                                       Sodium phosphonate 0-5%                                                       Trisodium citrate dihydrate 9-30%                                             Nitrilotrisodium acetate (NTA) 0-20%                                          Sodium perborate monohydrate 5-10%                                            Tetraacetylethylenediamine (TAED) 1-2%                                        Polyacrylate polymer 6-25%                                                    (e.g. maleic acid/acrylic acid co-                                            polymer)                                                                      Enzymes including modified enzymes 0.0001-0.5%                                Perfume 0.1-0.5%                                                              Water 5-10                                                                  ______________________________________                                    

    ______________________________________                                        3) POWDER AUTOMATIC DISHWASHING COMPOSITION                                   ______________________________________                                        Nonionic surfactant     0.5-2.0%                                                Sodium disilicate 25-40%                                                      Sodium citrate 30-55%                                                         Sodium carbonate 0-29%                                                        Sodium bicarbonate 0-20%                                                      Sodium perborate monohydrate 0-15%                                            Tetraacetylethylenediamine (TAED) 0-6%                                        Maleic acid/acrylic 0-5%                                                      acid copolymer                                                                Clay 1-3%                                                                     Poly(amino acids) 0-20%                                                       Sodium polyacrylate 0-8%                                                      Enzymes including modified enzymes 0.0001-0.5%                              ______________________________________                                    

    ______________________________________                                        4) POWDER AUTOMATIC DISHWASHING COMPOSITION                                   ______________________________________                                        Nonionic surfactant     1-2%                                                    Zeolite MAP 15-42%                                                            Sodium disilicate 30-34%                                                      Sodium citrate 0-12%                                                          Sodium carbonate 0-20%                                                        Sodium perborate monohydrate 7-15%                                            Tetraacetylethylenediamine (TAED) 0-3%                                        Polymer 0-4%                                                                  Maleic acid/acrylic acid copolymer 0-5%                                       Organic phosphonate 0-4%                                                      Clay 1-2%                                                                     Enzymes including modified enzymes 0.0001-0.5%                                Sodium sulphate Balance                                                     ______________________________________                                    

    ______________________________________                                        5) POWDER AUTOMATIC DISHWASHING COMPOSITION                                   ______________________________________                                        Nonionic surfactant     1-7%                                                    Sodium disilicate 18-30%                                                      Trisodium citrate 10-24%                                                      Sodium carbonate 12-20%                                                       Monopersulphate (2 KHSO.sub.5.KHSO.sub.4.K.sub.2 SO.sub.4) 15-21%                                    Bleach stabilizer 0.1-2%                               Maleic acid/acrylic acid copolymer 0-6%                                       Diethylenetriaminepentaacetate, 0-2.5%                                        pentasodium salt                                                              Enzymes including modified enzymes 0.0001-0.5%                                Sodium sulphate, water Balance                                              ______________________________________                                    

    ______________________________________                                        6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH                               CLEANING SURFACTANT SYSTEM                                                  ______________________________________                                        Nonionic surfactant       0-1.5%                                                Octadecyl dimethylamine N-oxide 0-5%                                          dihydrate                                                                     80:20 wt. C18/C16 blend of octadecyl 0-4%                                     dimethylamine N-oxide dihydrate and                                           hexadecyldimethyl amine N-oxide                                               dihydrate.                                                                    70:30 wt. C18/C16 blend of octadecyl 0-5%                                     bis (hydroxyethyl)amine N-oxide                                               anhydrous and hexadecyl bis                                                   (hydroxyethyl)amine N-oxide                                                   anhydrous                                                                     C.sub.13 -C.sub.15 alkyl ethoxysulfate with an 0-10%                          average degree of ethoxylation of 3                                           C.sub.12 -C.sub.15 alkyl ethoxysulfate with an 0-5%                           average degree of ethoxylation of 3                                           C.sub.13 -C.sub.15 ethoxylated alcohol with an 0-5%                           average degree of ethoxylation of                                             12                                                                            A blend of C.sub.12 -C.sub.15 ethoxylated alco- 0-6.5%                        hols with an average degree of                                                ethoxylation of 9                                                             A blend of C.sub.13 -C.sub.15 ethoxylated alco- 0-4%                          hols with an average degree of                                                ethoxylation of 30                                                            Sodium disilicate 0-33%                                                       Sodium tripolyphosphate 0-46%                                                 Sodium citrate 0-28%                                                          Citric acid 0-29%                                                             Sodium carbonate 0-20%                                                        Sodium perborate monohydrate 0-11.5%                                          Tetraacetylethylenediamine (TAED) 0-4%                                        Maleic acid/acrylic acid copolymer 0-7.5%                                     Sodium sulphate 0-12.5%                                                       Enzymes including modified enzymes 0.0001-0.5%                              ______________________________________                                    

    ______________________________________                                        7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING                                     COMPOSITION                                                                 ______________________________________                                        Liquid nonionic surfactant (e.g.                                                                      2.0-10.0%                                               alcohol ethoxylates)                                                          Alkali metal silicate 3.0-15.0%                                               Alkali metal phosphate 20.0-40.0%                                             Liquid carrier selected from higher 25.0-45.0%                                glycols, polyglycols, polyoxides,                                             glycolethers                                                                  Stabilizer (e.g. a partial ester of 0.5-7.0%                                  phosphoric acid and a C.sub.16 -C.sub.18                                      alkanol)                                                                      Foam suppressor (e.g. silicone) 0-1.5%                                        Enzymes including modified enzymes 0.0001-0.5%                              ______________________________________                                    

    ______________________________________                                        8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION                                 ______________________________________                                        Liquid nonionic surfactant (e.g.                                                                      2.0-10.0%                                               alcohol ethoxylates)                                                          Sodium silicate 3.0-15.0%                                                     Alkali metal carbonate 7.0-20.0%                                              Sodium citrate 0.0-1.5%                                                       Stabilizing system (e.g. mixtures 0.5-7.0%                                    of finely divided silicone and low                                            molecular weight dialkyl polyglycol                                           ethers)                                                                       Low molecule weight polyacrylate 5.0-15.0%                                    polymer                                                                       Clay gel thickener (e.g. bentonite) 0.0-10.0%                                 Hydroxypropyl cellulose polymer 0.0-0.6%                                      Enzymes including modified enzymes 0.0001-0.5%                                Liquid carrier selected from higher Balance                                   lycols, polyglycols, polyoxides and                                           glycol ethers                                                               ______________________________________                                    

    ______________________________________                                        9) THIXOTROPIC LIQUID AUTOMATIC                                                 DISHWASHING COMPOSITION                                                     ______________________________________                                        C.sub.12 -C.sub.14 fatty acid                                                                         0-0.5%                                                  Block co-polymer surfactant 1.5-15.0%                                         Sodium citrate 0-12%                                                          Sodium tripolyphosphate 0-15%                                                 Sodium carbonate 0-8%                                                         Aluminium tristearate 0-0.1%                                                  Sodium cumene sulphonate 0-1.7%                                               Polyacrylate thickener 1.32-2.5%                                              Sodium polyacrylate 2.4-6.0%                                                  Boric acid 0-4.0%                                                             Sodium formate 0-0.45%                                                        Calcium formate 0-0.2%                                                        Sodium n-decydiphenyl oxide 0-4.0%                                            disulphonate                                                                  Monoethanol amine (MEA) 0-1.86%                                               Sodium hydroxide (50%) 1.9-9.3%                                               1,2-Propanediol 0-9.4%                                                        Enzymes including modified enzymes 0.0001-0.5%                                Suds suppressor, dye, perfumes, Balance                                       water                                                                       ______________________________________                                    

    ______________________________________                                        10) LIQUID AUTOMATIC DISHWASHING COMPOSITION                                  ______________________________________                                        Alcohol ethoxylate      0-20%                                                   Fatty acid ester sulphonate 0-30%                                             Sodium dodecyl sulphate 0-20%                                                 Alkyl polyglycoside 0-21%                                                     Oleic acid 0-10%                                                              Sodium disilicate monohydrate 18-33%                                          Sodium citrate dihydrate 18-33%                                               Sodium stearate 0-2.5%                                                        Sodium perborate monohydrate 0-13%                                            Tetraacetylethylenediamine (TAED) 0-8%                                        Maleic acid/acrylic acid copolymer 4-8%                                       Enzymes including modified enzymes 0.0001-0.5%                              ______________________________________                                    

    ______________________________________                                        11) LIQUID AUTOMATIC DISHWASHING COMPOSITION                                    CONTAINING PROTECTED BLEACH PARTICLES                                       ______________________________________                                        Sodium silicate         5-10%                                                   Tetrapotassium pyrophosphate 15-25%                                           Sodium triphosphate 0-2%                                                      Potassium carbonate 4-8%                                                      Protected bleach particles, e.g. 5-10%                                        chlorine                                                                      Polymeric thickener 0.7-1.5%                                                  Potassium hydroxide 0-2%                                                      Enzymes including modified enzymes 0.0001-0.5%                                Water Balance                                                               ______________________________________                                    

11) Automatic dishwashing compositions as described in 1), 2) 3), 4), 6)and 10), wherein perborate is replaced by percarbonate.

12) Automatic dishwashing compositions as described in 1)-6) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in "Efficient manganesecatalysts for low-temperature bleaching", Nature, 369, (1994), p.637-639.

Examples of readily available products containing protease fordetergents include Alcalase®, Esperase®, Savinase® and Durazym® (allavailable from Novo Nordisk A/S); lipases for detergents includeLipolase® and Lipolase™ Ultra (available from Novo Nordisk A/S);cellulases for detergents such as Celluzyme®; α-amylases for detergentssuch as Termamyl®.

Personal Care Applications

The conjugated polypeptide of the invention is also of interest inconnection with personal care applications.

Proteases

Proteases are well-known active ingredients for cleaning of contactlenses. They hydrolyse the proteinaceous soil on the lens and therebymakes it soluble. Removal of the protein soil is essential for thewearing comfort.

Proteases are also effective ingredients in skin cleaning products,where they remove the upper layer of dead keratinaseous skin cells andthereby makes the skin look brighter and more fresh.

Proteases are also used in oral care products, especially for cleaningof dentures, but also in dentifrices.

Further, proteases are used in toiletries, bath and shower products,including shampoos, conditioners, lotions, creams, soap bars, toiletsoaps, and liquid soaps.

Lipases

Lipases can be applied for cosmetic use as active ingredients in skincleaning products and anti-acne products for removal of excessive skinlipids, and in bath and shower products such as creams and lotions asactive ingredients for skin care.

Lipases can also be used in hair cleaning products (e.g. shampoos) foreffective removal of sebum and other fatty material from the surface ofhair.

Lipases are also effective ingredients in products for cleaning ofcontact lenses, where they remove lipid deposits from the lens surface.

Oxidoreductases

The most common oxidoreductase for personal care purposes is an oxidase(usually glucose oxidase) with substrate (e.g. glucose) that ensuresproduction of H₂ O₂, which then will initiate the oxidation of forinstance SCN⁻ or I⁻ into antimicrobial reagents (SCNO⁻ or I₂) by aperoxidase (usually lactoperoxidase). This enzymatic complex is known innature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system in oral careproducts (mouth rinse, dentifrice, chewing gum) where it also can becombined with an amyloglucosidase to produce the glucose. These systemsare also known in cosmetic products for preservation.

Anti-microbial systems comprising the combination of an oxidase and aperoxidase are know in the cleaning of contact lenses.

Another application of oxidoreductases are oxidative hair dyeing usingoxidases, peroxidases and laccases.

Free radicals formed on the surface of the skin (and hair) known to beassociated with the ageing process of the skin (spoilage of the hair).

The free radicals activate chain reactions that leads to destruction offatty membranes, collagen, and cells. The application of free radicalscavengers such as Superoxide dismutase into cosmetics is well-known (R.L. Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) is also an oxidoreductase. It can beutilised for waving of hair (reduction and reoxidation of disulfidebonds in hair) and repair of spoiled hair (where the damage is mainlyreduction of existing disulfide bonds).

Carbohydrases

Plaque formed on the surface of teeth is composed mainly ofpolysaccharides. They stick to the surface of the teeth and themicroorganisms. The polysaccharides are mainly α-1,6 bound glucose(dextran) and α-1,3 bound glucose (mutan). The application of differenttypes of glucanases such as mutanase and dextranase helps hydrolysingthe sticky matrix of plaque, making it easier to remove by mechanicalaction.

Also other kinds of biofilm for instance the biofilm formed in lenscases can be removed by the action of glucanases.

Anti-microbial Polypeptides

Anti-microbial polypeptides have widespread applications such as forpreservation of cosmetic products, anti-acne products, deodorants andshampoos. Further such polypeptides may be use in contact lens products.

Food and Feed

Further conjugated enzymes or polypeptides with reduced allergenicityaccording to the invention may advantageously be used in themanufacturing of food and feed.

Proteases

The gluten in wheat flour is the essential ingredient responsible forthe ability of flour to be used in baked foodstuffs. Proteolytic enzymesare sometimes needed to modify the gluten phase of the dough, e.g. ahard wheat flour can be softened with a protease.

Neutrase® is a commercially available neutral metallo protease that canbe used to ensure a uniform dough quality and bread texture, and toimprove flavour. The gluten proteins is degraded either moderately ormore extensively to peptides, whereby close control is necessary inorder to avoid excessive softening of the dough.

Proteases are also used for modifying milk protein.

To coagulate casein in milk when producing cheese proteases such asrennet or chymosin may be used.

In the brewery industry proteases are used for brewing with unmaltedcereals and for controlling the nitrogen content.

In animal feed products proteases are used so to speak to expand theanimals digestion system.

Lipases

The application of lipase in the baking industry is rather new. Additionof lipase results in improved dough properties and an improvedbreadmaking quality in terms of larger volume, improved crumb structureand whiter crumb colour. The observed effect can be explained by amechanism where the lipase changes the interaction between gluten andsome lipids fragment during dough mixing. This results in an improvedgluten network.

The flavour development of blue roan cheeses (e.g. Danablue), certainItalian cheese types and other dairy products containing butter fat aredependent on the degradation of milk fat into free fatty acids. Lipasesmay be used for developing flavour in such products.

In the oil- and fat producing industry lipases are used e.g. to minimizethe amount of undesirable side-products, to modify fats byinteresterification, and to synthesis of esters.

Oxidoreductases

Further oxidoreductases with reduced allergenicity according to theinvention may advantageously be used in the manufacturing of food andfeed.

Several oxidoreductases are used for baking, glucose oxidase,lipoxygenase, peroxidase, catalase and combinations hereof.Traditionally, bakers strengthen gluten by adding ascorbic acid andpotassium bromate. Some oxidoreductases can be used to replace bromatein dough systems by oxidation of free sulfydryl units in glutenproteins. Hereby disulphide linkages are formed resulting in stronger,more elastic doughs with greater resistance.

Gluzyme™ (Novo Nordisk A/S) is a glucose oxidase preparation withcatalase activity that can be used to replace bromate. The doughstrengthen is measured as greater resistance to mechanical shock, betterover spring and larger loaf volume.

Carbohydrases

Flour has varying content of amylases leading to differences in thebaking quality. Addition of amylases can be necessary in order tostandardize the flour. Amylases and pentosanases generally provide sugarfor the yeast fermentation, improve the bread volume, retardretrogradation, and decrease the staling rate and stickiness thatresults from pentosan gums. Examples of carbohydrases is given below.

Certain maltogenic amylases can be used for prolonging the shelf life ofbread for two or more days without causing gumminess in the product.Selectively modifies the gelatinized starch by cleaving from thenon-reducing end of the starch molecules, low molecular wight sugars anddextrins. The starch is modified in such a way that retrogradation isless likely to occur. The produced low-molecular-weight sugars improvethe baked goods water retention capacity without creating theintermediate-length dextrins that result in gumminess in the finishedproduct. The enzyme is inactivated during bread baking, so it can beconsidered a processing aid which does not have to be declared on thelabel. Overdosing of Novamyl can almost be excluded.

The bread volume can be improved by fungal α-amylases which furtherprovide good and uniform structure of the bread crumb. Said α-amylasesare endoenzymes that produce maltose, dextrins and glucose. Cereal andsome bacterial α-amylases are inactivated at temperatures above thegelatinization temperature of starch, therefore when added to a wheatdough it results in a low bread volume and a sticky bread interior.Fungamyl has the advantage of being thermolabile and is inactivated justbelow the gelatinization temperature.

Enzyme preparations containing a number of pentosanase andhemi-cellulase activities can improve the handling and stability of thedough, and improves that freshness, the crumb structure and the volumeof the bread.

By hydrolysing the pentosans fraction in flour, it will lose a greatdeal of its water-binding capacity, and the water will then be availablefor starch and gluten. The gluten becomes more pliable and extensible,and the starch gelatinize more easily. Pentosanases can be used incombination with or as an alternative to emulsifiers.

Further carbohydrases are user for producing syrups from starch, whichare widely used in soft drinks, sweets, meat products, dairy products,bread products, ice cream, baby food, jam etc.

The conversion of starch is normally carried out three steps. First thestarch is liquefied, by the use of α-amylases. Maltodextrins, primaryconsisting of oligosaccharides and dextrins, are obtained.

The mixture is then treated with an amyloglucosidase for hydrolysing theoligosaccharides and dextrins into glucose. This way a sweeter productis obtained. If high maltose syrups are desired β-amylases alone or incombination with a pullulanase (de-branching enzyme) may be used.

The glucose mixture can be made even sweeter by isomerization tofructose. For this an immobilized glucose isomerase can be used.

In the sugar industry, it is common practice to speed up the break downof present starch in cane juices. Thereby the starch content in the rawsugar is reduced and filtration at the refinery facilitated.

Furthermore dextranases are used to break down dextran in raw sugarjuices and syrups.

In the alcohol industry α-amylases is advantageously being used forthinning of starch in distilling mashes.

In the brewing industry α-amylases is used for adjunct liquefaction.

In the dairy industry β-galactosidases (lactase) is used when producinglow lactose milk for persons suffering from lactose malabsorption.

When flavoured milk drinks are produced from lactase-treated milk, theaddition of sugar can be reduced without reducing the sweetness of theproduct.

In the production of condensed milk, lactose crystallization can beavoided by lactase treatment, and the risk of thickening caused bycasein coagulation in lactose crystals is thus reduced.

When producing ice cream made from lactase-treated milk (or whey) nolactose crystals will be formed and the defect, sandiness, will notoccur.

Further, xylanases are known to be used within a number of food/feedindustrial applications as described in WO 94/21785 (Novo Nordisk A/S).

α-amylases are used in the animal feed industry to be added tocereal-containing feed to improve the digestibility of starch.

Anti-microbial Polypeptides

Certain bacteriolytic enzymes may be used e.g. to wash carcasses in themeat packing industry (see U.S. Pat. No. 5,354,681 from Novo IndustriA/S)

Transferases

Transglutaminases with reduced allergenicity according to the inventionmay advantageously be used in the manufacturing of food and feed.

Transglutaminases has the ability to crosslinking protein.

This property can be used for gelling of aqueous phases containingproteins. This may be used for when producing of spreads (DK patentapplication no. 1071/84 from Novo Nordisk A/S).

Transglutaminases are being used for improvement of baking quality offlour e.g. by modifying wheat flour to be used in the preparation ofcakes with improved properties, such as improved taste, dent, mouth-feeland a higher volume (see JP 1-110147).

Further producing paste type food material e.g. used as fat substitutionin foods as ice cream, toppings, frozen desserts, mayonnaises and lowfat spreads (see WO 93/22930 from Novo Nordisk A/S).

Furthermore for preparation of gels for yoghurt, mousses, cheese,puddings, orange juice, from milk and milk-like products, and binding ofchopped meat product, improvement of taste and texture of food proteins(see WO 94/21120 and WO 94/21129 from Novo Nordisk A/S).

Phytases

Phytases of the invention may advantageously be used in themanufacturing of food, such as breakfast cereal, cake, sweets, drink,bread or soup etc., and animal feed.

Phytases may be used either for exploiting the phosphorus bound in thephytate/phytic acid present in vegetable protein sources or forexploiting the nutritionally important minerals bound in phytic acidcomplexes.

Microbial phytase may be added to feedstuff of monogastric animals inorder to avoid supplementing the feed with inorganic phosphorus (seeU.S. Pat. No. 3,297,548)

Further phytases may be used in soy processing. Soybean meal may containhigh levels of the anti-nutritional factor phytate which renders thisprotein source unsuitable for application in baby food and feed forfish, calves and other non-ruminants, since the phytate chelatesessential minerals present therein (see EP 0 420 358).

Also for baking purposes phytases may be used. Bread with better qualitycan be prepared by baking divided pieces of a dough containing wheatflour etc. and phytase (see JP-0-3076529-A)

A high phytase activity koji mold are known to be used for producingrefined sake (see JP-0-6070749-A).

Textile Applications

Proteases

Proteases are used for degumming and sand-washing of silk.

Lipases

Lipases are used for removing fatty matter containing hydrophobic esters(e.g. triglycerides) during the finishing of textiles (see e.g. WO93/13256 from Novo Nordisk A/S).

Oxidoreductases

In bleach clean-up of textiles catalases may serve to remove excesshydrogen peroxide.

Carbohydrases

Cellulolytic enzymes are widely used in the finishing of denim garmentsin order to provide a localized variation in the colour density of thefabric (Enzyme facilitated "stone wash").

Also cellulolytic enzymes find use in the bio-polishing process.Bio-Polishing is a specific treatment of the yarn surface which improvesfabric quality with respect to handle and appearance without loss offabric wettability. Bio-polishing may be obtained by applying the methoddescribed e.g. in WO 93/20278.

During the weaving of textiles, the threads are exposed to considerablemechanical strain. In order to prevent breaking, they are usuallyreinforced by coating (sizing) with a gelatinous substance (size). Themost common sizing agent is starch in native or modified form. A uniformand durable finishing can thus be obtained only after removal of thesize from the fabric, the so called desizing. Desizing of fabrics sizedwith a size containing starch or modified starch is preferablyfacilitated by use of amylolytic enzymes.

Oral and Dermal Pharmaceuticals

Proteases

Different combinations of highly purified proteases (e.g. Trypsin andChymotrypsin) are used in pharmaceuticals to be taken orally, and dermalpharmaceuticals for combating e.g inflammations, edemata and injuries.

Leather Production

Transferase

Transglutaminase is known to be used for casein finishing of leather byacting as a hardening agent (see WO 94/13839 from Novo Nordisk)

Hard Surface Cleaning

Cleaning of hard surfaces e.g. in the food industry is often difficult,as equipment used for producing dairies, meat, sea food products,beverages etc. often have a complicated shape. The use of surfactantcompositions in the form gels and foams comprising enzymes have shown tofacilitate and improve hard surface cleaning. Enzymes, whichadvantageously may be added in such surfactant compositions, are inparticular proteases, lipases, amylases and cellulases.

Such hard surface cleaning compositions comprising enzymes may alsoadvantageously be used in the transport sector, for instance for washingcars and for general vessel wash.

Finally the invention relates to the use of the conjugate of theinvention or a composition of the invention in products comprisingpolypeptides.

First of all the conjugate or compositions of the invention canadvantageously be used for personal care products, such as hair care andhair treatment products. This include products such as shampoo, balsam,hair conditioners, hair waving compositions, hair dyeing compositions,hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.

Further contemplated is oral care products such as dentifrice, mouthwashes, chewing gum.

Also contemplated is skin care products and cosmetics, such as skincream, skin milk, cleansing cream, cleansing lotion, cleansing milk,cold cream, cream soap, nourishing essence, skin lotion, milky lotion,calamine lotion, hand cream, powder soap, transparent soap, sun oil, sunscreen, shaving foam, shaving cream, baby oil lipstick, lip cream,creamy foundation, face powder, powder eye-shadow, powder, foundation,make-up base, essence powder, whitening powder.

Also for contact lenses hygiene products the conjugate of the inventioncan be used advantageously. Such products include contact lensescleaning and disinfection products.

The use for detergents such as washing powder, soap, soap bars, liquidsoap are also contemplated.

METHODS AND MATERIALS

Materials:

Enzyme Substrate

suc-AAPF-pNA(succinyl-Alanine-Alanine-Proline-Phenylalaninepara-nitroanilide. Sigmano. S-7388, M_(w) 624.6 g/mole

Dimethyl-casein (CM-casein) (sigma)

Glycerol tributyrate (Merck 1958)

Cellazyme-C® (Megazyme)

Carboxy methyl cellulose (Sigma)

Polymers:

Polyethylene glycol (PEG-35.000) from Fluka

Polyethylene glycol (PEG-5.000) activated by Tresyl chloride(2,2,2-triflouroethansulfonyl chloride) (Sigma, St. Louis, USA; M-3038)

mono-methoxypolyethylene glycol (mPEG-5.000) from Shearwater PolymersInc., USA.

mono-methoxypolyethylene glycol (mPEG-15.000) from Shearwater PolymersInc., USA.

mPEG-NH₂ -5.000 (Shearwater Polymers Inc., USA)

Enzymes:

Esperase® (available from Novo Nordisk A/S)

Subtilisin A (Novo Nordisk A/S)

Subtilisin Novo (Subtilisin BNP') (Novo Nordisk A/S) Lipolase®: Humicolalanuginosa lipase described in EP 305 216 (available from Novo NordiskA/S)

Lipolase® variant A: Lipolase® with the following mutations E87K/D254K(available from Novo Nordisk A/S)

Candida antarctica lipase B (available on request from Novo NordiskA/S).

Polyporus pinsitus laccase described in PCT/US95/07536 from Novo NordiskBiotech Inc.

Coprinus cinereus peroxidase (available from Novo Nordisk A/S onrequest).

Carezyme® (Novo Nordisk A/S)

Solutions:

Stop-solution (DMG-buffer)

Sodium Borate, borax (Sigma)

3,3-Dimethyl glutaric acid (Sigma) CaCl₂ (Sigma)

Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka) Tween20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184)1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka) N-Hydroxysuccinimide (Fluka art. 56480)) Phosgene (Fluka art. 79380)

Tracer Molecule:

biotinylated Mouse anti Rat IgE (Zymed, no. 03-9740)

Colouring Substrate: OPD: o-phenylene-diamine, (Kementec cat no. 4260)

Animals:

Brown Norway rats (from Charles River, DE)

Equipment:

XCEL II (Novex)

ELISA reader (UVmax, Molecular Devices)

HPLC (Waters)

PFLC (Pharmacia)

Superdex column, Mono-Q, Mono S from Pharmacia, SW.

Superdex-75 column (Pharmacia, SW)

SLT: Fotometer from SLT LabInstruments

Size-exclusion chromatograph (Spherogel TSK-G2000 SWG).

Size-exclusion chromatograph (Superdex 200, Pharmacia, SW)

Methods:

Protease Activity Using Casein as Substrate

The Esperase® activity determined using casein as the substrate isdescribed in "AF 219/1-GB (available from Novo Nordisk A/S).

Protease Activity Analysis with Suc-Ala-Ala-Pro-Phe-pNA:

Proteases especially chymotrypsin cleaves the bond between the peptide,and p-nitroaniline to give a visible yellow colour absorbing at 405 nm.

Buffer: e.g. Britton and Robinson buffer, pH 8.3 Substrate: 100 mgsuc-AAPF-pNA is dissolved into 1 ml dimethyl sulfoxide (DMSO). 100 μl ofthis is diluted into 10 ml with Britton and Robinson buffer.

Analysis: Substrate and protease solution is mixed and the absorbance ismonitored at 405 nm as a function of time and ABS₄₀₅ nm /min. Thetemperature should be controlled (20-50° C. depending on protease). Thisis a measure of the protease activity in the sample.

Lipase Activity:

The lipase activity was analysed as described in "AF 95/5 GB (availablefrom Novo Nordisk on request).

Oxidoreductase Activity

The Oxidoreductase activity determined using casein as the substrate isdescribed in "AF 219/1-GB (available from Novo Nordisk A/S).

Laccase Activity

Laccase activity is determined from the oxidation of syringaldazin underaerobic conditions. The violet colour produced is photometered at 530nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetatebuffer, pH 5.5, 30° C., 1 min. reaction time. 1 laccase unit (LACU) isthe amount of enzyme that catalyses the conversion of 1.0 μmolesyringaldazin per minute at these conditions.

The laccase activity of laccases is described in AF 239 GB which arehereby included as reference (available on request from Novo Nordisk).

Peroxidase Activity

The enzyme activity of the peroxidase is measured in PODU (peroxidaseunits). 1 PODU is the amount of enzymes that catalyses the conversion of1 μmol H₂ O₂ per minute in a system where2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonate], ABTS, is oxidized. Adetailed description of Novo Nordisk's analytical method is available onrequest (AF 279/1 GB).

Cellulase Activity

The enzymatic activity of Carezyme® was measured as release of blue dyefrom azurine-crosslinked HE-cellulose (Cellazyme-C®) The reaction wascarried out at 40° C. in 20 mM Na-phosphate pH 7 for 10 minutes. Releaseof dye was monitored by reading the absorbance at 595 nm in a UVmax®Elisa-reader. In addition, cellulytic activity was measured as describedin "EAL-SM-0373.01/01" (available from Novo Nordisk on request).

ELISA IgE Test System.

A three layer sandwich ELISA is used to determine relativeconcentrations of specific antibodies.

The immunizing molecule is used as coating antigen with 10 μg per ml and50 μl per well, in neutral phosphate buffer, incubated overnight at 4°C. All remaining binding spots on the well surface are blocked in 2%skim milk, 200 μl per well in phosphate buffer for at least 30 minutesat room temperature (RT). All seras to be tested with this antigen areadded at 50 μl per well to this plate using a 8-channel pipette indilution series from 10× diluted followed by 3-fold dilutions. Dilutionsare made i phosphate buffer with 0.5% skim milk and 0.05% Tween2o,incubated 2 hours on agitation platform at RT. The "tracer" molecule isbiotinylated Mouse anti Rat IgE 50 μl per well and diluted 2000× inphosphate buffer with 0.5% skim milk and 0.05% Tween 20, incubated 2hours on an agitation platform at RT. Control (blank) was iden-ticalsequence but without rat sera. 50 μl per well streptavidin horse raddishperoxidase, diluted 2000× was incubated 1 hour on an agitation platform.Colouring substrate at 50 μl per well is OPD (6 mg) and H₂ O₂ (4 μl of a30% solution) per 10 ml citrate buffer pH 5.2. The reaction is stoppedusing 100 μl per well 2 N H₂ SO₄. All readings on SLT at 486 nm and 620nm as reference. Data is calculated and presented in Lotus.

Intratracheal (IT) Stimulation of Rats

For IT administration of molecules disposable syringes with a 21/2" longmetal probe is used. This probe is instilled in the trachea (see FIG. 1)approximately 1 cm below the epiglotis (see FIG. 1), and 0.1 ml of asolution of the molecules is deposited. The animals are stimulated 4times, with 5 days between the last stimulation and exsanguination.Animals are Brown Norway rats, in groups of 4. Weight at time of startis more than 250 grams and at termination approximately 450 grams

Determination of the Molecular Weight

Electrophoretic separation of proteins was performed by standard methodsusing 4-20% gradient SDS poly acrylamide gels (Novex). Proteins weredetected by silver staining. The molecular weight was measured relativeto the mobility of Mark-12® wide range molecular weight standards fromNovex.

EXAMPLE 1

Activation of PEG 35.000 with Tresyl Chloride

The activation method is adapted from Nilson, K. et al., (1984), supra.All solvents used are Merck analytical grade.

4.0 g PEG 35.000 was dissolved in anhydrous dichloromethane (10 ml).Pyridine (0.25 ml) and Tresyl chloride (0.22 ml) was added. Uponstirring for 90 minutes at ambient temperature the obtained yellowmixture was evaporated to dryness and dissolved in hot ethanol (60 ml)and made acidic with HCl. A voluminous white precipitate was obtainedupon leaving the mixture overnight at -18° C. in a freezer. Theprecipitate was recovered by centrifugation at 400 g for 20 minutes andwashed repeatedly (6 times) with cold acidic ethanol (60 ml ethanol, 0.5ml concentrated HCl). The activated PEG-35.000 was recovered byevaporation of solvents until constant weight was obtained of anoff-white powder in a yield of 77%. The activated PEG-35.000 wascharacterised by melting point 59-61° C. and NMR analysis indicated30-35% tresylation.

In a scale up synthesis following the above method a yield of 96% withmelting point 59-60° C. light yellow flakes was obtained. The NMRanalysis showed above 40% tresylation.

EXAMPLE 2

Activation of mPEG 15.000 with Tresyl Chloride

mPEG 15.000 (10.0 g) was dissolved in dichloromethane (anhydrous 35 ml)of which 15 ml was distilled off to remove any trace of water. Aftercooling triethylamine (900 μl, 10 eqv.) and Tresyl chloride (350 μl, 5eqv.) was added below the surface. The solution turned light yellow andsome triethylamine hydrochloride precipitated. After 90 min the solutionwas poured into ether (250 ml) in a thin squirt and with stirring. After3 min. of stirring the light yellow precipitate was filtered, washedwith ether (20 ml) and dried to yield 11.3 g NMR showing 80-90%activation and significant amounts of HNEt₃ Cl. This was recrystallisedfrom ethyl acetate (325 ml) with warm filtration to remove most of thesalt. After slow cooling to room temperature the suspension was left inthe refrigerator for further crystallisation. Yielding 9.3 g (93%) ofwhite crystals. ¹ H-NMR (CDCl₃) δ 1.42 t (I=6.5 CH₃ i HNEt₃ Cl), 3.10 dq(I=4.6 CH₂ i HNEt₃ Cl), 3.38 s (I=2.6 CH₃ i OMe), 3.40* dd (I=4.5%, ¹³ Csatellite), 3.64 bs (I=1364 main peak), 3.89* dd (I=4.8%, ¹³ Csatellite), 4.24 q (J=9.0 Hz, I=1.8, CH₂ in tresyl), 4.53* dd (I=1.5 CH₂-O-Tresyl) Indicating 80-90% activation and only 6% (w/w) HNEt₃ Cl.

EXAMPLE 3

Activation of mPEG 15.000 with N-succinimidyl Carbonate

mPEG 15.000 was suspended in toluene (4 ml/g of mPEG) 20% was distilledoff at normal pressure to dry the reactants azeotropically.Dichloromethane (dry 1 ml/g mPEG) was added when the solution was cooledto 30° C. and phosgene in toluene (1.93 M 5 mole/mole mPEG) was addedand mixture stirred at room temperature over night. The mixture wasevaporated to dryness and the desired product was obtained as waxylumps.

After evaporation dichloromethane and toluene (1:2, dry 3 ml/g mPEG) wasadded to redissolve the white solid. N-Hydroxy succinimide (2 mole/molemPEG.) was added as a solid and then triethylamine (1.1 mole/mole mPEG).The mixture was stirred for 3 hours. initially unclear, then clear andending with a small precipitate. The mixture was evaporated to drynessand recrystallised from ethyl acetate (10 ml) with warm filtration toremove salts and insoluble traces. The blank liquid was left for slowcooling at ambient temperature for 16 h and then in the refrigeratorover night. The white precipitate was filtered and washed with a littlecold ethyl acetate and dried to yield 98% (w/w). NMR Indicating 80-90%activation and 5% (w/w) HNEt₃ Cl. ¹ H-NMR for mPEG 15000 (CDCl₃) δ 1.42t (I=4.8 CH₃ i HNEt₃ Cl), 2.84 s (I=3.7 succinimide), 3.10 dq (I=3.4 CH₂i HNEt₃ Cl), 3.38 s (I=2.7 CH₃ i OMe), 3.40* dd (I=4.5%, ¹³ Csatellite), 3.64 bs (I=1364 main peak), 3.89* dd (I=4.8%, ¹³ Csatellite), 4.47 dd (I=1.8, CH₂ in PEG). No change was seen afterstorrage in desiccator at 22° C. for 4 months.

EXAMPLE 4

Conjugation of mPEG 5.000 with N-succinimidvl Carbonate

Activation of mPEG 5.000 with N-succinimidyl carbonate was performed asdescribed in Example 3.

EXAMPLE 5

Conjuration of Protease with Tresyl Chloride-activated PEG-35.000

A mixture (7.5 ml) of 70 mg of highly purified Esperase® and 855 mg ofactivated PEG 35.000 prepared according to example 1 was incubated in0.1 M Na-Borate, pH 9.2, at ambient temperature overnight using magneticstirring. The conjugation was terminated by addition of ethanol amine(0.01 ml).

The resulting Esperase® PEG 35.000 conjugate was purified bysize-exclusion chromatography by HPLC using a Superdex-75 column.

Compared to the parent enzyme the conjugate held 68% residual enzymeactivity in peptide assay using suc-AAPF-pNA as substrate and 39%residual enzyme activity the assay using casein as substrate.

EXAMPLE 6

Using the same procedure and chemicals as describe in the above examples1 and 6, with approximately 10 times surplus of activated PEG 35.000,e.g. 42 mg of Esperase® and 480 mg of activated PEG residual activitiesrelative to the parent enzyme are 80% using the suc-AAPF-pNA-substrateand 46% with casein as substrate.

EXAMPLE 7

Conjugation of 3Protease with Activated mPEG 5.000

200 mg of Subtilisin Novo was incubated in 50 mM NaBorate, pH 10, with1.8 g of activated mPEG 5.000 with N-succinimidyl carbonate (preparedaccording to Example 4), in a final volume of 20 ml. The reaction wascarried out at ambient temperature using magnetic stirring. Reactiontime was 1 hour. The reaction was stopped by adding DMG buffer to afinal concentration of 5 mM dimethyl glutarate, 1 mM CaCl₂ and 50 mMborate, pH 5.0.

The molecular weight of the obtained derivative was approximately 100kDa, corresponding to 12 moles of PEG attached per mole Subtilisin Novo.

Compared to the parent enzyme, residual activity was close to 100%towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide) and64% towards CM-casein.

EXAMPLE 8

Conjugation of Protease with Activated mPEG 15.000

200 mg of Subtilisin Novo was incubated in 50 mM NaBorate pH 10 with 5.5g of activated mPEG 15.000 N-succinimidyl carbonate (prepared accordingto Example 3), in a final volume of 20 ml. The reaction was carried outat ambient temperature using magnetic stirring. Reaction time was 1hour. The reaction was stopped by adding DMG buffer to a finalconcentration of 5 mM dimethyl glutarate, 1 mM CaCl₂ and 50 mM borate,pH 5.0.

The molecular weight of the obtained derivative was above 200 kDa,corresponding to 12 moles of PEG attached per mole Subtilisin Novo.

Compared to the parent enzyme, residual activity was close to 100%towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide) and71% towards CM-casein.

EXAMPLE 9

Conjugation of Lipase with Tresyl Chloride Activated PEG 35.000

A mixture (3.4 ml) of 40 mg of highly purified Lipolase® and 440 mg ofactivated PEG 35.000 prepared according to example 1 was incubated in0.1 M Na-Borate, pH 9.2, at ambient temperature over night usingmagnetic stirring. The conjugation was terminated by addition of ethanolamine (0.01 ml).

The resulting Lipolase®-PEG 35.000 conjugate was purified bysize-exclusion chromatography by HPLC using a Superdex-75 column.

Compared to the parent lipase the conjugate held 56% residual enzymeactivity using glycerol tributyrate as substrate.

EXAMPLE 10

Conjugation of Lipase with Tresyl Chloride Activated PEG 5.000

A mixture (2 ml) of 25 mg of highly purified Lipolase® and 363 mg ofTresyl chloride activated PEG 5.000 was incubated in 0.1 M Na-Borate, pH9.2, at ambient temperature over night using magnetic stirring. Theconjugation was terminated by addition of ethanol amine (0.01 ml).

The resulting Lipolase®-PEG 5.000 conjugated was purified bysize-exclusion chromatography by HPLC using a Superdex-75 column.

Compared to the parent enzyme the conjugate held 48% residual enzymeactivity using glycerol tributyrate as substrate.

EXAMPLE 11

Conjugation of Lipase with Tresyl Chloride Activated mPEG 15.000

25 mg of Candida antarctica lipase B in 25 ml of 0.1 M borate, 1 M NaCl,pH 9.2, was incubated with 2.82 gram of mPEG 15.000 activated withTresyl chloride according to Example 2 for 3 hours at ambienttemperature. The reaction was stopped by addition of 1 ml 2M Glycine andthe derivative purified by size-exclusion chromatography (SpherogelTSK-G2000 SWG).

Relative to the parent Candida antarctica lipase B the derivativeretained some 83% residual enzyme activity using glycerol tributyrate assubstrate.

EXAMPLE 12

Conjugation of Lipase with Tresyl Chloride Activated mPEG 15.000

27 mg of Lipolase®variant A in 6 ml of 0.1 M borate, 0.8M NaCl, pH 9.2was incubated with 417.4 mg of mPEG 15.000 activated with TresylChloride according to Example 2 for 3 hours at ambient temperature. Thereaction was stopped by addition of 139 μl 2M Glycine and the derivativepurified by size-exclusion chromatography (Spherogel TSK-G2000 SWG).

Relative to the parent Lipolase® variant the derivative retained some36% residual enzyme activity using glycerol tributyrate as substrate.

EXAMPLE 13 Conjugation of Lipase with Tresyl Chloride Activated mPEG15.000

170 mg of Lipolase® in 17 ml of 0.1 M borate, 0.8 M NaCl, pH 9.2 wasincubated with 2.19 g of mPEG 15.000 activated according to Example 2for 3 hours at ambient temperature. The reaction was stopped by additionof 730 μA 2M Glycine and the derivative purified by size-exclusionchromatography (Spherogel TSK-G2000 SWG).

Relative to the parent Lipolase® the derivative retained some 64%residual enzyme activity using glycerol tributyrate as substrate.

EXAMPLE 14

Conjugation of Lipase with N-succinimidyl Carbonate Activated mPEG15.000

100 mg of Lipolase® in 9 ml of 0.1 M borate, 1M NaCl, pH 9.2 wasincubated with 2.576 g of mPEG-15000 activated with N-succinimidylcarbonateaccording to Example 3 for 3 hours at ambient temperature. Thereaction was stopped by addition of 859 μl 2M Glycine and the derivativepurified by size-exclusion chromatography (Spherogel TSK-G2000 SWG)

Relative to the parent Lipolase® the derivative retained some 60%residual enzyme activity using glycerol tributyrate as substrate.

The molecular weight of the obtained conjugate was determined to beabout 150 kDa using SDS-PAGE.

EXAMPLE 15

Carbodiimide Mediated Conjugation of Lipase with mPEG-NH₂ -5.000

0.4 mg of Lipolase® in 5 ml of 50 MM MES buffer, 0.2M NaCl, pH 5.0 wasincubated with 400 mg of mPEG-NH₂ -5.000, and 125 mg1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 3 hours atambient temperature. The derivative was purified by size-exclusionchromatography (Superdex 200, Pharmacia).

Relative to the parent Lipolase® the derivative retained about 1%residual enzyme activity using glycerol tributyrate as substrate.

EXAMPLE 16

Conjugation of Laccase with N-succinimidyl Carbonate Activated mPEG15.000

100 mg of Polyporus pintitus laccase in 10 ml of 0.1 M borate, 0.5 MNaCl, pH 9.2 was incubated with 1.155 g of mPEG 15.000 activated withN-succinimidyl carbonate according to Example 3 for 2 hours at ambienttemperature. The reaction was stopped by addition of 200 μl 2 M Glycineand the derivative purified by size-exclusion chromatography (Superdex200) and dialysed towards 50 mM borate, pH 9.0.

Molecular weights were determined by SDS-PAGE to be in the range of 150to 200 kDa.

Relative to the parent Laccase the derivative retained some 55% residualenzyme activity.

EXAMPLE 17

Conjuration of Coprinus cinereus Peroxidase with N-succinimidylCarbonate Activated mPEG 15.000

75 mg of peroxidase in 7.5 ml of 0.1 M borate, 0.5 NaCl, pH 9.2 asincubated with 1.579 g of mPEG 15.000 activated with N-succinimidylcarbonate according to Example 3 for 2 hours at ambient temperature. Thereaction was stopped by addition of 200 μl 2M Glycine and the derivativepurified by size-exclusion chromatography (Superdex 200) and dialysedtowards 0.1 M Na-phosphate, pH 7.0.

Molecular weights are determined by SDS-PAGE to be in the range of 150to 200 kDa.

Relative to the parent peroxidase the derivative retained some 79%residual enzyme activity.

EXAMPLE 18

Conjugation of Cellulase with Activated Tresyl Chloride Activated PEG5.000

27.2 mg of Carezyme® was incubated in 50 mM NaCarbonate buffer pH 8.5with 115.2 mg of Tresyl chloride activated PEG 5.000. The reaction wascarried out at ambient temperature using magnetic stirring. Reactiontime was 1 hour.

The molecular weight of the derivatives (assessed as the relativemobility of denatured proteins in SDS polyacrylamide gels) was 53-63kDa, corresponding 2-4 moles of PEG attached per mole Carezyme®.

Compared to an internal standard (a highly purified preparation of theparent enzyme), the derivative held 63% residual activity.

EXAMPLE 19

Conjugation of Substrate Protected Cellulose with Activated mPEG 5.000

To shield the active site of the enzyme during mPEG conjugation, theenzyme was diluted in a carboxymethyl cellulose solution (0.5% W/V) tothe same final buffer and protein concentration as in Example 3 (4 mlNaCarbonate pH 8.5, 6.8 mg/ml Carezyme® and 115.2 mg of N-succinimidylcarbonate activated mPEG 5.000). The reaction was conducted at 4° C. for1 hour.

As in Example 18, the degree of derivatization was evaluated by SDSelectrophoresis using 4-20% gradient polyacrylamide gels, followed bysilver staining. The apparent mass of the derivative was 53-63 kDa,corresponding 2-4 moles of PEG attached per mole Carezyme®.

The catalytic activity of the derivative was 90% of that of the parentenzyme.

EXAMPLE 20

Conjugation of Product Stabilized Cellulase with Activated mPEG 5.000

To protect the active site of Carezyme®, cellobiose (Sigma, C7292) wasadded to a final concentration of 80 mM, corresponding to a molar excessof 5.000 fold. The conjugation of N-succinimidyl carbonate activatedmPEG 5.000 with Carezyme® in the presence of cellobiose, and thecharacterization of the products, was carried out as described in above.The apparent mass of the derivative was 53-63 kDa, Corresponding 2-4moles of PEG attached per mole Carezyme®. The derivative held 100%residual activity.

EXAMPLE 21

Conjugation of Cellulase with N-succinimidyl Carbonate Activated mPEG15.000

20 mg of Carezyme® in 1 ml of 0.1 M borate, 1 mM CaCl₂, pH 10.0, wasincubated with 54.3 mg of mPEG 15.000 activated with N-succinimidylcarbonate according to Example 3 for 3 hours at ambient temperature. Thereaction was stopped by addition of 18 μl 2 M Glycine and the derivativepurified by size-exclusion chromatography (Superdex 200).

Relative to the parent Carezyme® the modified Carezyme® retained some66% residual enzyme activity.

EXAMPLE 22

Rat Intratrachaeal (IT) Trails

Brown Norway rats (BN) was stimulated intratrachaeally (IT) withmodified Subtilisin Novo, Lipolase®, Polyporus pinsitus laccase andcarezyme®, respectively, all enzymes conjugation with N-succinimidylcarbonate activated mPEG 15.000 as described in the examples above, andthe corresponding parent enzymes as controls.

Sera from immunized animals were tested in a specific IgE ELISA(described above) to elucidate whether the molecules had penetrated thelung epithelias and activated the immune response system giving rise toa specific IgE respons (See FIGS. 2, 3, 4 and 5).

As can be seen from the figures the response of the rats exposedintratracheally with the modified enzyme is reduced in comparison torats having been exposed intratracheally with the parent enzymes.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. Accordingly, the scope of the invention is to beconstrued in accordance with the substance defined by the followingclaims.

What is claimed is:
 1. A composition comprising(a) a modifiedmetalloendopeptidase, wherein said metalloendopeptidase is conjugated toa polymer with a molecular weight in the range of 1 kDa to 60 kDa,wherein said modified metalloendopeptidase exhibits reduced respiratoryallergenicity relative to an unmodified counterpart and (b) a substancefor industrial application which renders the composition unsuitable forpharmaceutical use.
 2. The composition according to claim 1, whereinsaid polymer is selected from the group consisting of polyalkyleneoxides (PAO), polyvinyl alcohol (PVA), poly-carboxylates,poly-(vinylpyrolidone), poly-D,L-amino acids, dextrans, hydrolysates ofchitosan, starches, guar gum, pullulan, inulin, xanthan gum,carrageenin, pectin, and alginic acid hydrolysates.
 3. The compositionaccording to claim 1, wherein said polymer is a polyalkylene oxideselected from the group consisting of polyethylene glycols (PEG),methoxypolyethylene glycols (mPEG) and polypropylene glycols,PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),Star-PEGs, and branched PEGs.
 4. The composition according to claim 1,wherein said polymer has a molecular weight (M_(r)) between 2 kDa and 35kDa.
 5. The composition according to claim 1, wherein saidmetalloendopeptidase is conjugated with from 1 to 25 polymer molecules.6. A composition comprising(a) a modified enzyme selected from the groupconsisting of Subtilisin Novo, Subtilisin DY, subtilisin BPN',subtilisin amylosacchariticus, subtilisin 168, subtilisinmesentericopeptidase, subtilisin Carlsberg, subtilisin 309, thermitase,aqualysin, Bacillus protease PB92, proteinase K, Protease TW7, andProtease TW3, wherein said enzyme is conjugated to a polymer with amolecular weight in the range of 1 kDa to 60 kDa, wherein said modifiedenzyme exhibits reduced respiratory allergenicity relative to anunmodified enzyme and (b) a substance for industrial application whichrenders the composition unsuitable for pharmaceutical use.
 7. Thecomposition according to claim 6, wherein said polymer is selected fromthe group consisting of polyalkylene oxides (PAO), polyvinyl alcohol(PVA), poly-carboxylates, poly-(vinylpyrolidone), poly-D,L-amino acids,dextrans, hydrolysates of chitosan, starches, guar gum, pullulan,inulin, xanthan gum, carrageenin, pectin, and alginic acid hydrolysates.8. The composition according to claim 6, wherein said polymer is apolyalkylene oxide selected from the group consisting of polyethyleneglycols (PEG), methoxypolyethylene glycols (mPEG) and polypropyleneglycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole(CDI-PEG), Star-PEGs, and branched PEGs.
 9. The composition according toclaim 6, wherein said polymer has a molecular weight (M_(r)) between 2kDa and 35 kDa.
 10. The composition according to claim 6, wherein saidpolypeptide is conjugated with from 1 to 25 polymer molecules.
 11. Acomposition comprising(a) a modified enzyme selected from the groupconsisting of amylases, xylanases, dextranases, α-galactatases,glucoamylases, and maltogenic amylase, wherein said enzyme is conjugatedto a polymer with a molecular weight in the range of 1 kDa to 60 kDa,wherein said modified enzyme exhibits reduced respiratory allergenicityrelative to an unmodified enzyme and (b) a substance for industrialapplication which renders the composition unsuitable for pharmaceuticaluse.
 12. The composition according to claim 11, wherein said polymer isselected from the group consisting of polyalkylene oxides (PAO),polyvinyl alcohol (PVA), poly-carboxylates, poly-(vinylpyrolidone),poly-D,L-amino acids, dextrans, hydrolysates of chitosan, starches, guargum, pullulan, inulin, xanthan gum, carrageenin, pectin, and alginicacid hydrolysates.
 13. The composition according to claim 11, whereinsaid polymer is a polyalkylene oxide selected from the group consistingof polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) andpolypropylene glycols, PEG-glycidyl ethers (Epox-PEG),PEG-oxycarbonylimidazole (CDI-PEG), Star-PEGs, and branched PEGs. 14.The composition according to claim 11, wherein said polymer has amolecular weight (M_(r)) between 2 kDa and 35 kDa.
 15. The compositionaccording to claim 11, wherein said polypeptide is conjugated with from1 to 25 polymer molecules.
 16. The composition according to claim 11,wherein the substance for industrial application is selected from thegroup consisting of a personal care product, an oral care product, askin care product, a cosmetic, a contact lenses hygiene product, adetergent, an oral or dermal pharmaceutical, an agrochemical, food orfeed, a baking product, a product for processing textiles, and acomposition for cleaning hard surfaces.
 17. The composition according toclaim 11, wherein the substance for industrial application is adetergent selected from the group consisting of washing powder, liquiddetergent, dishwashing detergent, and soap.
 18. A compositioncomprising(a) a modified subtilisin 147, wherein said subtilisin isconjugated to a polymer selected from the group of polyalkylene oxides(PAO), polyvinyl alcohol (PVA), poly-carboxylates,poly-(vinylpyrolidone), and poly-D,L-amino acids with a molecular weightin the range of 1 kDa to 60 kDa, wherein said modified subtilisinexhibits reduced respiratory allergenicity relative to an unmodifiedenzyme and (b) a substance for industrial application which renders thecomposition unsuitable for pharmaceutical use.
 19. The compositionaccording to claim 18, wherein said polyalkylene oxide is selected fromthe group consisting of polyethylene glycols (PEG), methoxypolyethyleneglycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers(Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), Star-PEGs, and branchedPEGs.
 20. The composition according to claim 18, wherein said polymerhas a molecular weight (M_(r)) between 2 kDa and 35 kDa.
 21. Thecomposition according to claim 18, wherein said polypeptide isconjugated with from 1 to 25 polymer molecules.